Calcium-Sensing Receptors of Human Astrocyte-Neuron Teams: Amyloid-ß-Driven Mediators and Therapeutic Targets of Alzheimer's Disease.

It is generally assumed that the neuropathology of sporadic (late-onset or nonfamilial) Alzheimer's disease (AD) is driven by the overproduction and spreading of first Amyloid-ßx-42 (Aß42) and later hyperphosphorylated (hp)-Tau oligomeric "infectious seeds". Hitherto, only neurons were held to make and spread both oligomer types; astrocytes would just remove debris. However, we have recently shown that exogenous fibrillar or soluble Aß peptides specifically bind and activate the Ca(2+)-sensing receptors (CaSRs) of untransformed human cortical adult astrocytes and postnatal neurons cultured in vitro driving them to produce, accrue, and secrete surplus endogenous Aß42. While the Aß-exposed neurons start dying, astrocytes survive and keep oversecreting Aß42, nitric oxide (NO), and vascular endothelial growth factor (VEGF)-A. Thus astrocytes help neurons' demise. Moreover, we have found that a highly selective allosteric CaSR agonist ("calcimimetic"), NPS R-568, mimics the just mentioned neurotoxic actions triggered by Aß●CaSR signaling. Contrariwise, and most important, NPS 2143, a highly selective allosteric CaSR antagonist ("calcilytic"), fully suppresses all the Aß●CaSR signaling-driven noxious actions. Altogether our findings suggest that the progression of AD neuropathology is promoted by unceasingly repeating cycles of accruing exogenous Aß42 oligomers interacting with the CaSRs of swelling numbers of astrocyte-neuron teams thereby recruiting them to overrelease additional Aß42 oligomers, VEGF-A, and NO. Calcilytics would beneficially break such Aß/CaSR-driven vicious cycles and hence halt or at least slow the otherwise unstoppable spreading of AD neuropathology.

Effects of epidermal growth factor/urogastrone and associated pancreatic hormones on mitotic cycle phases and proliferation kinetics of neonatal rat hepatocytes in primary culture.

Low doses (10(-14)--10(-11) M) of epidermal growth factor (EGF)/urogastrone and/or equimolar mixtures of glucagon and insulin administered to 4-day-old primary neonatal rat liver cultures stimulated both DNA synthesis and proliferation of hepatocytes within 24 h. The precise roles played in this process by EGF and the two pancreatic hormones were investigated by constructing curves of the fraction of labeled hepatocytes in mitosis and studying the dilution of incorporated [3H]thymidine with time. The results indicate that EGF both activates and then promotes prereplicative development of hepatocytes, while glucagon and insulin only promote prereplicative development.

Effect of glucagon and insulin on the growth of neonatal rat hepatocytes in primary tissue culture.

Commercial (bovine-porcine) glucagon added in a single dose between 10(-12) and 10(-7) M to neonatal rat hepatocytes in primary cultures with subsequent incubation for 20-24 h, stimulated their entry into the DNA synthesis phase as revealed by [3H]thymidine-labeling and radioautography; about 14 h of incubation was required before an effect was observed. Commercial (bovine) insulin at doses between 10(-11) and 10(-7) M apparently stimulated the entry of hepatocytes into S phase. However, insulin's effect, which needed 20 h for induction, was due to the release of a wave of synchronized hepatocytes from an earlier produced block near the G1/S boundary of their growth-division cycle. Equimolar mixtures of glucagon with insulin from 10(-15)-10(-7) M increased the fraction of hepatocytes synthesizing DNA first at 4-8 h, and then at 20-24 h. Effective doses of glucagon, insulin, and glucagon plus insulin also increased the entry of hepatocytes into mitosis, as found after a 4-h incubation with colchicine (0.1 mM). Withholding inactivated fetal bovine serum from the growth medium for 24 h did not change the mitotic activity either of the untreated or of the glucagon- and glucagon plus insulin-stimulated hepatocytes, but it increased the proliferogenic effect of bovine insulin. Highly purified crystalline (porcine) glucagon, insulin, and glucagon plus insulin also stimulated the growth of hepatocytes in the presence or absence of serum. Finally, equimolar (10(-14) M) mixtures of glucagon with (Bu)2cGMP and of insulin with (Bu)2cAMP increased the hepatocytic replication as efficiently as did glucagon plus insulin at the same dose. The present results show that glucagon and insulin are synergistic, intracycle regulators of the growth of neonatal rat hepatocytes. They also suggest that cyclic necleotides may mediate at least partly the hepatotropic effects of the pancreatic hormones.

Cross-linked trimers of bovine ribonuclease A: activity on double-stranded RNA and antitumor action.

Trimers of bovine pancreatic RNase A were obtained by cross-linking native RNase A with dimethyl suberimidate. They degrade double-stranded RNA more efficiently than dimers and monomers of RNase A, and display significant cytotoxic and/or cytostatic actions against C4-I cells (a human cell line derived from squamous carcinoma of the uterus cervix). On the same cell line cross-linked dimers of RNase A appear to be ineffective.

TPA and cycloheximide modulate the activation of NF-kappa B and the induction and stability of nitric oxide synthase transcript in primary neonatal rat hepatocytes.

12-O-Tetradecanoylphorbol 13-acetate (TPA) elicited a transient increase in the transcription of the inducible nitric oxide synthase (iNOS) gene coupled with a shortening of the half-life of its mRNA in primary neonatal rat hepatocytes. These effects of TPA were preceded by a surge in nuclear translocation of the transcription factor NF-kappa B, and followed by a mounting accumulation of NO-2 in the growth medium. Even cycloheximide (CHX) added by itself elicited an early, sustained activation of NF-kappa B followed by an intense induction of iNOS gene expression, irrespective of what degree of protein synthesis inhibition was brought about by the several concentrations tested. When given together, TPA and CHX exerted additive effects on hepatocellular iNOS mRNA levels. These results suggest the likelihood of an ordered sequence of events by which an activated NF-kappa B mediates the induction of iNOS gene expression in TPA- and/or CHX-treated primary hepatocytes.

Morphology, growth, and gene expression in five newly isolated murine hepatocellular tumor cell lines.

Five murine hepatocellular tumor cell lines (HepM-1-5) were isolated and grown in a synthetic medium added with hormones, growth factors and/or serum. The morphology of these lines ranged from a nearly homogeneous epithelial-like shape (HepM-2) to a stromal appearance (HepM-1). The remaining lines displayed a mixed morphology. For their proliferation all of the cell lines retained a clear dependence on the extracellular calcium level and hormonal and/or serum growth factors and, rather homogeneously, they did not express the albumin, alpha-fetoprotein (with the exception of HepM-2 cells), tyrosine aminotransferase, and ornithine transcarbamylase genes, whereas they all exhibited discrete levels of the ornithine aminotransferase mRNA. Only HepM-3 and HepM-5 lines expressed the procollagen type I gene.

Donor's and recipient's antigen presenting cells co-operatively enhance the allo-reactive proliferation of peripheral blood lymphocytes.

Antigen-presenting cells (APC) play important roles in the allo-reactive proliferation of T cells in vitro and in the allo-graft rejection in vivo. This work was aimed at establishing whether a co-operation of any kind occurs between donor's and recipient's APC during allo-reactions, and whether certain cell surface molecules, such as beta 2-microglobulin (beta 2m) and human leucocyte antigens (HLA), are involved in such an interaction. The data herein reported show that the allo-reactive proliferation of peripheral blood lymphocytes (PBL) is markedly intensified by a positive co-operation between donor's and recipient's APC. Such a synergistic co-stimulatory action can be significantly hindered by a monoclonal antibody aimed against beta 2m while being not changed by monoclonal antibodies binding to either HLA-A-B-C or HLA-DR molecules. Hence, our preliminary results indicate that a positive interaction between donor's and recipient's APC may be of importance for allo-graft rejection.

Prostaglandins of the F series are extremely powerful growth factors for primary neonatal rat hepatocytes.

At low concentrations (i.e., 10(-12)-10(-9) mol/1), PGF1 alpha and PGF2 alpha very intensely stimulated both the DNA-synthetic and mitotic activities of hepatocytes in 4-day-old primary cultures of neonatal rat liver. DNA replication was more intensely enhanced by PGF2 alpha than by PGF1 alpha, whereas mitotic activity was nearly equally affected by the two prostaglandins. On the whole, the growth-promoting activity of PGF1 alpha used by itself or in equimolar mixtures with other prostaglandins (e.g., A1, E1, etc.) mimicked that of arachidonic acid we previously reported (1). On a molar basis, PGF2 alpha by itself stimulated hepatocytes' DNA synthesis more powerfully than arachidonate did, and when used in equimolar mixtures with other prostaglandins was at least as potent as arachidonic acid. These observations establish prostaglandins of the F series as quite powerful commitment factors and, though by a lesser degree, also intracycle regulators for neonatal rat hepatocytes in primary culture. However, the understanding of the role(s) of prostaglandins of F and other series in the physiological control of hepatocytes' proliferative activation must await the clarification of their interaction(s) with other arachidonate derivative(s) and polypeptide growth factor(s) which also may be involved in the process.

The calcium-dependent stimulation of the proliferation of neonatal rat hepatocytes by imidazole and indomethacin.

Low concentrations (e.g. 10(-12) and 10(-11) mol/l) of imidazole and indomethacin strongly stimulated DNA synthesis and mitosis of hepatocytes in 4-day-old primary cultures of neonatal rat liver. These agents seem to have acted by inducing quiescent hepatocytes to begin cycling rather than by affecting already cycling cells, because they did not shorten the total cell cycle time. Neither compound stimulated DNA synthesis by hepatocytes cultured in low (0.010 mol/l) calcium medium. Nevertheless, hepatocytes in calcium-deficient medium must have been mitogenically activated by these compounds and, hence, been able to reach a late stage of prereplicative development because they did initiate DNA synthesis very soon after the addition of calcium.

Effect of adenosine 3',5'-cyclic monophosphate on the RNA and DNA synthesis and cell proliferation of rat hepatocytes in primary culture: a radioautographic study.

The effect of a 24-h treatment with various doses (from 1.5-10-minus 8 to 3.0-10-minus 3 M) of adenosine 3',5'-cyclic monophospahte (cAMP) on morphometric parameters, [5--3H]uridine radioactivity concentration (URC), [methyl--3H]thymidine [Me--3H]-Tr) labelling index per hour (L.I./h) and per cent mitotic index (M.I.%) of young rat differentiated hepatocytes in primary tissue culture were investigated by morphometric and radioautographic methods. In such cells cAMP was found to induce: (1) a reduction of the apparent surface area (ASA) of total nucleoli, karyoplasm and cytoplasm; (2) significant increases in URC of all the subcellular compartments at all the dosages employed (only cAMP at 1.5-10-minus 8 M did not change karyoplasmic and cytoplasmic URC values); (3) marked increments in [Me--3H]Tdr L.I./h and M.I.% from the lowest dose up to 1.5-10-minus 4 M; at higher doses the L.I./h and M.I.% were less stimulated or approached control values. In cultured rat hepatocytes, adenosine-5'-phosphate (5'-AMP) (1.5-10-minus 4 M per 24 h) increased the karyoplasmic and total cell ASA, the lone total nucleolar URC and both the L.I./h and M.I.%. However, these metabolic effects were significantly less intense than those elicited by isomolar cAMP. Theophylline (Theo) (5.5-10-minus 5 M per 24 h) reduced the in vitro rat hepatocyte total nucleolar ASA but affected neither other morphometric nor any of the URC values. The same dose of Theo plus cAMP (1.5-10-minus M) had no morphometric effect but significantly increased the URC values of all primary rat hepatocyte compartments. Actinomycin D (DAct) (0.1 mug/ml per 24 h) plus cAMP (1.5-10-minus 4 M) decreased the cultured rat hepatocyte total nucleolar ASA but enlarged that of karyoplasm and cytoplasm and, further, markedly curtailed all the compartmental URC values. These data support the hypothesis that cAMP amplified the template activity of the liver chromatin and accelerates the flow of differentiated primary young rat hepatocytes into the various stages of the mitotic cell cycle.

The calcium-dependence of the stimulation of neonatal rat hepatocyte DNA synthesis and division by epidermal growth factor, glucagon and insulin.

A low concentration (10(-11) mol/l) of epidermal growth factor (EGF) and/or an equimolar (10(-14) mol/l) mixture of glucagon and insulin stimulated DNA synthesis in hepatocytes in 4-day-old primary cultures of neonatal rat liver. EGF seems to have acted by inducing quiescent hepatocytes to begin cycling, while the glucagon-insulin combination seems to have acted mainly by shortening the cell cycle time. Incubation in low calcium medium blocked untreated hepatocytes in the G1 phase of their cycle and prevented EGF and the glucagon-insulin mixture from stimulating DNA synthesis. Nevertheless, hepatocytes in calcium-deficient medium did respond to these agents, as they reached a late stage of prereplicative development before being blocked: in fact, they initiated DNA synthesis soon after the addition of calcium. EGF, but not the glucagon-insulin combination, also enabled the already cycling hepatocytes (but not the newly activated ones) to overcome the block imposed by the extracellular calcium deficiency after a delay of several hours.

The primary tissue culture of human adrenocortical Conn's adenomata. I. The synergistic stimulation of adenomatous cell growth by purine cyclic nucleotides and by ACTH1-24 and angiotensin II.

Primary cultures of dissociated parenchymal cells from Conn's adenomata causing primary hyperaldosteronism were successfully set up by a method previously used with normal adult human and rat adrenocortical tissue. In such cultures the adenomatous cells largely prevailed (making up 87% of the whole cell population), could survive for lengthy terms (at least up to 30 days), and were endowed with a spontaneous, discrete capability to proliferate. The de novo RNA- and DNA-synthetic and mitotic activities of Conn's cells were markedly stimulated in cultures exposed between 16 and 21 to daily doses of exogenous cyclic AMP, either alone or in equimolar association with cyclic GMP. A significantly weaker, though still prominent enhancement of adenomatous cell growth was elicited also by daily administrations of an equimolar mixture of ACTH1-24 and angiotensin II. In contrast, little stimulation or inhibition of growth or no effect at all could be observed when cyclic GMP, ACTH1-24, and angiotensin II were respectively administered, each by itself.

Prolongation of survival of alloskin grafts with no concurrent general suppression of the burned patient's immune system: a preliminary clinical investigation.

The adequate management of burns is the requisite to save the patient's life, to prevent the onset of secondary infections and to obtain healing with acceptable cosmetic results. Previous data have shown that the use of alloskin grafts in the acute phase of burns yields the best clinical results. Unfortunately, this approach is curbed by the patient's immune response which induces the rejection of the allografts. In this report we endeavoured to solve this problem by ways that would not imply the general suppression of the patient's immune system. Thus, we can now report that a longer survival of alloskin grafts can be induced in situ by pretreating the alloskin samples to be grafted with both an anti-beta 2-microglobulin monoclonal antibody (beta 2mAb) and irradiation with ultraviolet-C light (UVC). The advantage of our novel approach is that it does not need any concomitant administration of immunosuppressive agent(s) to the burned patients. In this study, we followed five severely burned patients grafted with alloskin samples pretreated with either beta 2mAb or beta 2mAb plus UVC irradiation. In comparison with untreated alloskin samples from the same source grafted in parallel, the mean survival time (MST) of the beta 2mAb-pretreated alloskin specimens increased by 35 per cent (i.e. from 18.3 +/- 3.2 days to 24.7 +/- 5.5 days) in three patients. Moreover, the MST of the alloskin grafts pretreated with both beta 2mAb and UVC irradiation was lengthened by 100 per cent (i.e. from 18.5 +/- 4.5 days to 37.0 +/- 8.0 days) in the remaining two patients. These preliminary results lend credence to the view that local immune suppression could be achieved in situ by our approach via: (1) the impairment of the proper functions of the HLA-class I antigen by the bound beta 2mAb; and (2) the inhibition of immune reactions taking place inside the alloskin grafts by some immunosuppressive signal(s) generated by the UVC-pretreated alloepidermal cells. Hence, our approach stands as an exciting and useful alternative to the otherwise well-known, deleterious general suppression of the burned patient's immune system.

An investigation into the mechanisms by which human dermis does not significantly contribute to the rejection of allo-skin grafts.

The dermis is an important element in skin substitutes and in allo- or xeno-skin grafts. However, the reason(s) why dermis does not significantly induce the immune rejection reaction in vivo remain(s) hitherto unknown. To clarify the mechanisms underlying this phenomenon, we undertook the evaluation of: (i) the response of the peripheral blood mononuclear cells (PBM) to isolated allo-dermal cells or to pieces of or to whole allo-dermis, (ii) the migration and homing of the PBM inside allo-dermis or split thickness allo-skin, (iii) the distribution of the ICAM-1 protein within skin, and (iv) the features expressed by the PBM that migrate into allo-skin. The results herein presented show that (1) the isolated allo-dermal cells had the highest and the whole allo-dermis the lowest capacity to initiate the reactive proliferation of the PBM in vitro; (2) in an allo-skin/PBM co-culture model, most of the PBM slowly, yet preferentially, migrated to and homed inside the allo-epidermal compartment, instead of staying in the allo-dermis; (3) under the conditions employed, rather little ICAM-1 could be immunohistochemically detected within the epidermis, conversely, both the dermal cells and the dermal matrix were ICAM-1 positive; and (4) most of the PBM migrating into the allo-skin pieces expressed either the CD18 or the CD19 or the CD8 molecule, yet very few of them exhibited the LFA-1-antigen, and none of them were found to be CD4 positive.2+Therefore, we conclude that because

A novel approach to extend the survival of skin xenografts without entailing general immunosuppression or systemic toxicity.

The feasibility of using antigenically disguised skin xenotransplants to cover extensive burns for a suitable time lag without administering immunosuppressive drugs was tested experimentally. Pieces of human skin that had been preincubated for 3 h at 37 degrees C with either mouse anti-human beta 2-microglobulin monoclonal antibody (beta 2m-McAb) or PBS (controls) were grafted onto the backs of immunologically competent Swiss mice, and the time required for their rejection or substitution by normal autogenous skin was determined. Thus, it was found that the beta 2m-McAb-pretreated xenografts had a significantly longer mean survival time than the control grafts. An even longer skin xenograft MST was obtained when beta 2m-McAb was repeatedly injected, at weekly intervals, just beneath the transplants. Parallel immunohistochemical studies showed that the beta 2m-McAb entered the grafts and was bound to its targets both in epidermis and dermis. Moreover, a small amount of beta 2m-McAb administered at the outset significantly hindered the reactive proliferation of primed mouse spleen cells cultured in the presence of human epidermal cells. Finally, neither toxic effects nor a weakening of immune competence were elicited by repeated intraperitoneal injections of beta 2m-McAb. Therefore, it seems expedient to propose the use of beta 2m-McAb to delay the rejection of skin xenografts as this antibody harmlessly prevents, wholly or in part, the activation of the recipients' lymphocytes. This would positively aid any patient urgently needing xenograft cover of extensive burns.

Primary tissue culture of human adrenocortical Conn's adenomata. Bromocriptine as a possible agonist-antagonist of angiotensin at the cellular level.

Angiotensin II-induced the hypertrophy of the cytoplasmic compartment and significantly increased (5-3H)uridine incorporation into RNA species by Conn's human adult adenomatous cells in primary tissue culture. On its own, bromocriptine, while enlarging only the nucleolar compartment, also intensely stimulated the incorporation of (5-3H)uridine into RNA species by the cultured adrenocortical adenomatous cells. However, an equimolar mixture of angiotensin II and bromocriptine was totally ineffective, eliciting no change in cellular morphometry or isotope incorporation with respect to the control specimens run in parallel. The present findings support the view that bromocriptine can influence the metabolism of Conn's cells directly at the cellular level by acting as an agonist-antagonist of angiotensin.

Inhibitors of ADP-ribosyl transferase suppress the mitogenic actions exerted by tumour promoters, but not those evoked by peptide mitogens, in primary neonatal rat hepatocytes.

A significant stimulation of the 24-h (between day 4 and 5 in vitro) new DNA synthetic activity was elicited in primary neonatal rat hepatocytes kept in low-calcium (0.01 mmol/l) HiWoBa2000 synthetic medium by the addition of a single dose (10(-10) mol/l) of each of several tumour promoters [i.e. 12-O-tetradecanoylphorbol-13-acetate (TPA), phenobarbital, nafenopin, saccharin, teleocidin, benzoyl peroxide butylhydroxytoluene (BHT), dichlorodiphenyltrichloroethane (DDT), lindane, clofibrate and melittin]. Even hormones [e.g. epidermal growth factor (EGF), glucagon and insulin at 10(-10) mol/l] and EGF-like acting drugs (i.e. imidazole and indomethacin, at 10(-11) mol/l) similarly enhanced with respect to untreated controls the 24-h flow into S phase of the primary hepatocytes on condition, however, that the cells were incubated in a high- (i.e. 1.8 mmol/l) and not a low-calcium HiWoBa2000 medium. Xenobiotics, peptide mitogens and EGF-like acting drugs also enhanced the in vitro hepatocellular mitotic activity. The growth-stimulatory effects of the aforementioned eleven tumour promoters were entirely suppressed by the simultaneous addition to the growth medium of a fully effective dose (10(-4) - 10(-3) mol/l) of agents, such as 3-aminobenzamide (3-ABA), 3-methoxybenzamide (3-MBA) or nicotinamide (NA), that are known to inhibit the activity of ADP-ribosyl transferase (ADPRT). However, under the same conditions these inhibitors hampered neither the basal DNA synthetic and mitotic activities of spontaneously cycling hepatocytes nor the stimulation of the hepatocellular growth processes evoked by peptide mitogens and EGF-like acting drugs. Quantitative autoradiographic investigations showed that the incorporation of the ADP-ribose precursor and ADPRT substrate [3H]NAD into nuclear macromolecules of gently digitonin-permeabilized hepatocytes was negligible in the untreated cultures, whereas it was strikingly and nearly steadily increased by a 2-, 8- and 24-h exposure to a fully mitogenic dose (10(-10) mol/l) of TPA, thereby revealing that an early, significant and roughly steady activation of the nuclear ADPRT had taken place in the phorbol ester-treated liver parenchymal cells. The simultaneous addition of 3-ABA (10(-4) mol/l) not only fully checked the mitogenic effects of TPA, but even suppressed about two-thirds of the TPA-elicited nuclear incorporation of [3H]NAD by the permeabilized hepatocytes, thus showing that a significant curtailment of the TPA-activated ADPRT did occur is association with the abatement of the mitogenic effects of TPA by this inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)