Therapies for Prevention and Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia associated with a progressive neurodegenerative disorder, with a prevalence of 44 million people throughout the world in 2015, and this figure is estimated to double by 2050. This disease is characterized by blood-brain barrier disruption, oxidative stress, mitochondrial impairment, neuroinflammation, and hypometabolism; it is related to amyloid-ß peptide accumulation and tau hyperphosphorylation as well as a decrease in acetylcholine levels and a reduction of cerebral blood flow. Obesity is a major risk factor for AD, because it induces adipokine dysregulation, which consists of the release of the proinflammatory adipokines and decreased anti-inflammatory adipokines, among other processes. The pharmacological treatments for AD can be divided into two categories: symptomatic treatments such as acetylcholinesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists and etiology-based treatments such as secretase inhibitors, amyloid binders, and tau therapies. Strategies for prevention of AD through nonpharmacological treatments are associated with lifestyle interventions such as exercise, mental challenges, and socialization as well as caloric restriction and a healthy diet. AD is an important health issue on which all people should be informed so that prevention strategies that minimize the risk of its development may be implemented.

Immune growth hormone (GH): localization of GH and GH mRNA in the bursa of Fabricius.

Expression of growth hormone (GH) and GH receptor (GHR) genes in the bursa of Fabricius of chickens suggests that it is an autocrine/paracrine site of GH production and action. The cellular localization of GH and GH mRNA within the bursa was the focus of this study. GH mRNA was expressed mainly in the cortex, comprised of lymphocyte progenitor cells, but was lacking in the medulla where lymphocytes mature. In contrast, more GH immunoreactivity (GH-IR) was present in the medulla than in the cortex. In non-stromal tissues, GH-IR and GH mRNA were primarily in lymphocytes, and also in macrophage-like cells and secretory dendritic cells. In stromal tissues, GH mRNA, GH and GHR were expressed in cells near the connective tissue (CT) between follicles and below the outer serosa. In contrast, GH (but not GH mRNA or GHR), was present in cells of the interfollicular epithelium (IFE), the follicle-associated epithelium (FAE) and the interstitial corticoepithelium. This mismatch may reflect dynamic temporal changes in GH translation. Co-expression of GHR-IR, GH-IR, GH mRNA and IgG was found in immature lymphoid cells near the cortex and in IgG-IR CT cells, suggesting an autocrine/paracrine role for bursal GH in B-cell differentiation.

Heterogeneity of growth hormone immunoreactivity in lymphoid tissues and changes during ontogeny in domestic fowl.

Growth hormone (GH) expression is not confined to the pituitary and occurs in many extrapituitary tissues. Here, we describe the presence of GH-like moieties in chicken lymphoid tissues and particularly in the bursa of Fabricius. GH-immunoreactivity (GH-IR), determined by ELISA, was found in thymus, spleen, and in bursa of young chickens, but at concentrations <1% of those in the pituitary gland. Although the GH concentration in the spleen and bursa was approximately 0.82 and 0.23% of that in the pituitary at 9-weeks of age, because of their greater mass, the total GH content in the spleen, bursa, and in thymus were 236, 5.18, and 31.5%, respectively, of that in the pituitary gland. This GH-IR was associated with several proteins of different molecular size, as in the pituitary gland, when analyzed by SDS-PAGE under reducing conditions. While most of the GH-IR in the pituitary was associated with the 26 kDa monomer (40%), the putatively glycosylated 29 kDa variant (16%), the 52 kDa dimer (14%) and the 15 kDa submonomeric isoform (16%), GH-IR in the lymphoid tissues was primarily associated (27-36%) with a 17 kDa moiety, although bands of 14, 26, 29, 32, 37, 40, and 52 kDa were also identified in these tissues. The heterogeneity pattern and relative abundance of bursal GH-IR bands were determined during development between embryonic day 13 (ED13) and 9-weeks of age. The relative proportion of the 17 kDa GH-like band was higher (45-58%) in posthatched birds than in the 15 and 18-day old embryos (21 and 19%, respectively). The 26 kDa isoform was minimally present in embryos (<4% of total GH-IR) but in posthatched chicks it increased to 12-20%. Conversely, while GH-IR of 37, 40, and 45 kDa were abundantly present in embryonic bursa ( approximately 30% at ED13 and approximately 52-55% at ED15 and ED18, respectively), in neonatal chicks and juveniles they accounted for less than 5%. These ontogenic changes were comparable to those previously reported for similar GH-IR proteins in the chicken testis during development. In summary, these results demonstrate age-related and tissue-specific changes in the content and composition of GH in immune tissues of the chicken, in which GH is likely to be an autocrine or paracrine regulator.

Chicken growth hormone: further characterization and ontogenic changes of an N-glycosylated isoform in the anterior pituitary gland.

Glycosylation is one of the post-translational modifications that growth hormone (GH) can undergo. This has been reported for human, rat, mouse, pig, chicken and buffalo GH. The nature and significance of GH glycosylation remains to be elucidated. This present study further characterizes glycosylated chicken GH (G-cGH) and examines changes in the pituitary concentration of G-cGH during embryonic development and post hatching growth. G-cGH was purified from chicken pituitaries by affinity chromatography (Concanavalin A-Sepharose and monoclonal antibody bound to Sepharose). Immunoreactive G-cGH has a MW of 26 kDa or 29 kDa as determined by SDS-PAGE, respectively, under non-reducing and reducing conditions. Evidence that it is N-glycosylated comes from its susceptibility to peptide N-glycosidase F, and its resistance to O-glycosidase. Based on the ability of G-cGH to bind Concanavalin A or wheat germ agglutinin but not other lectins and its susceptibility to peptide N-glycosidase F, a hybrid or biantennary type glycopeptide (GlcNac2, Man) structure is proposed. Some G-cGH can be observed in the pituitary at most ages examined (from 15-day embryo to adult). Moreover, electron microscopy revealed the presence of both immuno-reactive GH and Concanavalin A-reactive sites in the same secretory granules in the somatotrope. There were marked changes in the level and relative proportion of G-cGH in the pituitary gland during development and growth, the proportion of G-cGH rising during late embryonic development (e.g., between 15 and 18 days of development) and with further increases between 9 weeks and 15 weeks old. G-cGH was able to bind to chicken liver membrane preparations with less affinity than non-glycosylated monomer; on the other hand, however, G-cGH stimulated cell proliferation on Nb2 lymphoma bioassay whereas the non-glycosylated monomer was uncapable to do it.

Growth hormone in the male reproductive tract of the chicken: heterogeneity and changes during ontogeny and maturation.

Growth hormone (GH) gene expression is not confined to pituitary somatotrophs and occurs in many extrapituitary tissues. In this study, we describe the presence of GH moieties in the chicken testis. GH-immunoreactivity (GH-IR), determined by ELISA, was found in the testis of immature and mature chickens, but at concentrations <1% of those in the pituitary gland. The immunoassayable GH concentration in the testis was unchanged between 4 and 66 weeks of age, and approximately 10-fold higher than that at 1-week of age and 25-fold higher than that in 1-day-old chicks and perinatal (embryonic day 18) embryos. This immunoreactivity was associated with several proteins of different molecular size, as in the pituitary gland, when analyzed by SDS-PAGE under reducing conditions. However, while most of the GH-IR in the pituitary ( approximately 40 and 15%, respectively) is associated with monomer (26 kDa) or dimer (52 kDa) GH moieties GH-IR in the testis is primarily (30-50%) associated with a 17 kDa moiety. GH bands between 32 and 45 kDa are also relatively more abundant in the testis than in the pituitary. During ontogeny the relative abundance of a 14 kDa GH and 40 kDa GH moieties in the testis significantly declined, whereas the relative abundance of the 17 and 45 kDa moieties increased with advancing age. In adult birds, GH-IR was widespread and intense in the seminiferous tubules. Although the GH-IR was not present in the basal compartment of Sertoli cells, nor in spermatogonia and primary spermatocytes, it was abundantly present in secondary spermatocytes and spermatids in the luminal compartments of the tubules as well as in some surrounding myocytes and interstitial cells. In summary, immunoreactive GH moieties are present in the chicken testis but at concentrations far less than in the pituitary. Age-related changes in the relative abundance of testicular GH variants may be related to local (autocrine/paracrine) actions of testicular GH. The localization of GH in spermatocytes and spermatids suggests hitherto unsuspected roles in gamete development.

Characterization of a bioactive 15 kDa fragment produced by proteolytic cleavage of chicken growth hormone.

There is evidence for a cleaved form of GH in the chicken pituitary gland. A 25 kDa band of immunoreactive-(ir-)GH, as well as the 22 kDa monomeric form and some oligomeric forms were observed when purified GH or fresh pituitary extract were subjected to SDS-PAGE under nonreducing conditions. Under reducing conditions, the 25 kDa ir-GH was no longer observed, being replaced by a 15 kDa band, consistent with reduction of the disulfide bridges of the cleaved form. The type of protease involved was investigated using exogenous proteases and monomeric cGH. Cleaved forms of chicken GH were generated by thrombin or collagenase. The site of cleavage was found in position Arg133-Gly134 as revealed by sequencing the fragments produced. The NH2-terminal sequence of 40 amino acid residues in the 15 kDa form was identical to that of the rcGH and analysis of the remaining 7 kDa fragment showed an exact identity with positions 134-140 of cGH structure. The thrombin cleaved GH and the 15 kDa form showed reduced activity (0.8% and 0.5% of GH, respectively) in a radioreceptor assay employing a chicken liver membrane preparation. However, this fragment had a clear bioactivity in an angiogenic bioassay and was capable to inhibit the activity of deiodinase type III in the chicken liver.

Evidence for a histocompatibility locus probably linked to but distinct from Mls and H-25.

BALB/c (Mlsb) and BALB.D2-Mlsa strains of mice, both H-2d, are congenic and differ for the Mls locus (and linked genes) located on chromosome 1. The BALB.D2-Mlsa strain was obtained by introducing the Mlsa allele of DBA/2 mice into BALB/c mice. In previous studies we showed that BALB.D2-Mlsa recipients reject, relatively rapidly, all skin grafts from BALB/c donors. We and other groups have questioned whether the rejections observed were indeed due to the incompatibility for Mlsb products or for products of a histocompatibility (non-H-2) locus linked to, but distinct from, Mlsb. To answer this question, several hybrids carrying either Mlsa or Mlsb in various genetic contexts were grafted with skin from Mls-compatible BALB/c or BALB.D2-Mlsa donors; in the genetic combinations selected, any rejection which might occur would reflect the effects of a non-Mls incompatibility between BALB/c and BALB.D2-Mlsa strains. In certain of the donor-recipient combinations studied, the skin grafts were tolerated for greater than 200 days, but a relatively rapid rejection of BALB/c skin grafts was observed in (B10.D2 x BALB.D2-Mlsa)F1 and (B10.BR x BALB. D2-Mlsa)F1 hybrid recipients. These results indicated that in addition to Mls, the BALB/c and BALB.D2-Mlsa strains differ for at least one other non-H-2 histocompatibility locus. The possible involvement of H-25 was then investigated. Indeed, disparity for H-25, which maps on chromosome 1 close to Mls, can induce relatively rapid skin graft rejection. The H-25 allele of the DBA/2 strain has not been defined: we considered, therefore, that BALB/c and DBA/2 could be disparate at the H-25 locus, and that H-25 (transmitted by DBA/2 to the BALB.D2-Mlsa strain, together with the Mlsa allele) could be responsible for the skin graft rejection we observed. Our results showed, however, that DBA/2, BALB/c and BALB.D2-Mlsa strains of mice all share the H-25c allele; they therefore ruled out a role for H-25 incompatibility in the skin graft rejections we observed, and indicated that these rejections are due to the effects of a yet undefined histocompatibility locus (locus 'x'), probably linked to, but distinct from, the Mls locus. Further experiments showed that the histocompatibility effect of locus 'x' cumulates with that exhibited by Mlsb (or by a putative histocompatibility locus linked to Mlsb).

Abrogation of lethal graft-versus-host reaction directed against non-H-2 antigens: role of Mlsa and K/I region antigens in the induction of unresponsiveness by alloimmunization.

A graft-versus-host reaction (GVHR) directed against DBA/2 non-H-2 antigens alone can be induced by grafting B10.D2 bone marrow and spleen cells intravenously to heavily irradiated, H-2d compatible (DBA/2 X B10.D2)F1 adult mice. Under the experimental conditions used, only 0-10% of recipients survive, but the survival is greatly increased by donor alloimmunization, a few days prior to grafting, against host-specific (DBA/2) non-H-2 antigens and non-specific (foreign) H-2 antigens. The increased survival is mediated by alloimmunization-activated suppressor cells which can decrease the intensity of the immune reaction developed by normal B10.D2 cells both in vivo (GVHR) and in vitro (proliferative response measured in mixed lymphocyte culture, MLC). The present experiments were designed to explore the antigenic requirements for inducing suppression. The results showed that in GVHR the protective effect induced by donor alloimmunization against the specific non-H-2 antigens, which leads to 70-80% survival, is due primarily, if not entirely, to immunization against Mlsa antigens. Results of MLC experiments confirmed this conclusion, showing that immunization against Mlsa antigens is sufficient to account for the suppressive effect induced by the specific immunization. In addition, they indicated that the non-specific protective effect induced by donor alloimmunization against foreign H-2 antigens, which leads to 20-30% survival, is due to immunization against antigens encoded by the K and/or I region(s) of the H-2 complex; immunization against D region encoded antigens alone has no effect.

Parameters involved in the induction and abrogation of the lethal graft-versus-host reaction directed against non-H-2 antigens.

The grafting of cells from donors incompatible for non-H-2 antigens alone can lead to GvHR mortality in up to 100% of lethally irradiated adult recipients. GvHR severity correlates with the number of mature immunocompetent cells present in the bone marrow inoculum. Histologic and clinical manifestations of GvHR observed in these mice differ from those seen when GvHR is induced across an H-2 barrier. The number of non-H-2 genes capable of influencing GvHR mortality is probably great, and their effects may vary as a function of sex. The non-H-2 genes influence GvHR mortality mainly via their interactions, the consequences of which are complex and can result in either cumulative or suppressive effects. GvHR mortality is considerably reduced by donor immunization, shortly before grafting, against host-specific non-H-2 antigens; and it is virtually abrogated by an additional immunization of the donors against nonspecific (foreign) H-2 antigens. Three weeks after grafting, these "protected" mice are easily distinguishable from those undergoing lethal GvHR, as assessed by both clinical appearance and histologic examination; in contrast, they are nearly indistinguishable from control mice grafted with syngeneic cells. However, depending upon the conditions used for the immunization, an additional immunization against nonspecific H-2 antigens can lead to acceleration rather than suppression of GvHR mortality; this phenomenon is not seen, under the same experimental conditions, after immunization against specific non-H-2 antigens alone. It is therefore suggested that a "second signal" provided by an additional nonspecific stimulus can potentiate either the establishment of specific suppression or the activation of a secondary ("positive") response. Suppressive effects of the specific and nonspecific immunizations are cumulative, and both treatments activate suppressor cells. The intensity of suppression induced by both specific and nonspecific immunizations is antigen dose-dependent. At equivalent antigen doses the specific immunization is considerably more effective than the nonspecific immunization, and is detectable after injection of as few as 2.5 X 10(5) cells. In both cases, irradiation of the immunizing cells abolishes the suppression induced by the lower cell doses tested, while it merely decreases the intensity of the suppression induced by the higher cell doses tested. The impairment of suppression after irradiation of the immunizing cells is not attributable to a modification of their homing pattern, but to the fact that proliferation of the immunizing cells, which leads to an augmentation of the antigen dose, is abolished by irradiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Soluble Mlsa antigens: stimulatory effect in vitro versus suppressive effect in vivo.

Using a pair of congenic strains of mice differing only at the Mls haplotype (Mls locus and closely linked genes), BALB/c (Mlsb) and BALB.D2-Mlsa, we have compared the in vitro proliferative responses of Mlsb lymphocytes to Mlsa antigens presented on either lymph node cells (LNC) or peritoneal adherent cells (PAC). Results showed that Mlsa-PAC are stronger stimulators than Mlsa-LNC, and furthermore, that the supernatant from Mlsa-PAC may be effective in eliciting a lymphocyte proliferative response. The proliferation in response to PAC supernatant is partially due to activation by nonspecific factor(s); however, the response in the presence of Mlsa incompatible PAC supernatant is about three times greater than the response obtained in the presence of syngeneic Mlsb-PAC supernatant, suggesting an additional stimulation by soluble Mlsa antigens. Contrasting with the ability of PAC-supernatant to stimulate a primary proliferative response in vitro, the in vivo immunization of Mlsb mice with Mlsa-PAC supernatant abrogates the specific proliferative response in subsequent one-way mixed lymphocyte cultures. This abrogation of the specific response is comparable to that observed after immunization with intact Mlsa peritoneal or spleen cells, although in the latter case the anti-H-2 proliferative response is also decreased, regardless of whether the H-2 incompatible stimulating cells express an additional incompatibility for Mlsa. The proliferation of untreated, but not of Mlsa-immunized BALB/c LNC, is stronger in cultures with DBA/2 stimulating cells (incompatible for Mlsa and other non-H-2 antigens) than in cultures with BALB.D2-Mlsa cells (incompatible for Mlsa alone), and is comparable in intensity to that activated by H-2 incompatibility. We conclude that Mlsa antigens are more efficiently recognized by unprimed helper T cells when presented on PAC than when presented on LNC. In the primary proliferative response, the effects of Mlsa and other non-H-2 antigens may be cumulative. In vivo immunization against Mlsa antigens results in suppression of the specific proliferative response and, to a certain extent, of the nonspecific proliferative response (directed against both H-2 and other non-H-2 antigens). Since Mlsa antigens are obtainable in soluble form, their physico-chemical purification can now be envisaged.

Strong histocompatibility and cell-mediated cytotoxic effects of a single Mls difference demonstrated using a new congenic mouse strain.

In vivo and in vitro effects of incompatibility at the Mls locus have been studied utilizing a recently created congenic mouse strain. Results obtained with skin grafts were compared to those obtained in the mixed lymphocyte reaction (MLR) and cell-mediated cytotoxicity assays. The in vitro responsiveness of cells from skin-grafted mice was compared to that of cells from corresponding ungrafted mice. The results showed that: (a) Mlsa, strongly stimulatory in primary MLR, has a weak effect on skin graft rejection; specific in vivo preimmunization against Mlsa increases and accelerates the rejection of skin grafts, but abrogates the responsiveness in MLR; and (b) incompatibility for Mlsb, nonstimulatory in primary MLR, induces relatively rapid rejection of 100% of skin grafts; this rejection is dramatically accelerated by specific in vivo preimmunization and is followed by activation of helper and cytotoxic cells. Results obtained in the cell-mediated cytotoxicity assay suggest that the recognition of Mlsb determinants is H-2-restricted. Finally, the rejection of skin grafts incompatible for numerous non-H-2 loci is delayed by an additional incompatibility for Mlsb, suggesting that Mlsb decreases the response to other non-H-2 antigens, thus acting as a suppressor and/or competitor antigen. We conclude that, in contrast with previous findings, Mls incompatibility may have a strong effect on skin graft rejection, depending on the allelic combination involved, and, after in vivo immunization, Mlsb activates cell-mediated proliferative and cytotoxic responses and definitely is not "silent". The importance of the histocompatibility effects of Mls determinants and the variety of its biological functions are much in favor of the existence of a polymorphic and complex system capable of activating different cell subsets.

Changes in albumin uptake during the lifespan of human fibroblasts in vitro.

It has been suggested that a deterioration of cell membrane functions in cell populations with a limited lifespan in vitro could explain the loss of division potential either through changes in permeability of in cell attachment. We analyzed membrane function measuring the uptake of iodinated human serum albumin (125ISA) at different passages during the lifespan in vitro of human adult lung fibroblasts. Monolayers were incubated with 100microng/ml 125ISA. One and sixty minutes later, cultures were washed and the cell bound radioactivity was determined; these values correspond respectively to adsorption and net uptake. Our results show a significant increased uptake of albumin by aging cells. The changes in cell permeability, however, are apparent only late during the life span. Old cultures were also more susceptible to the stimulatory action of polyornithine (PLO) on protein uptake. Results obtained with PLO on young cells showed that the cell takes up more albumin when there is membrane danage leading to leakage of proteins. These findings suggest that the increased uptake of albumin and the suceptibility to PLO are signs of membrane damage in cells that have reached the end of their lifespans. In that case, an increased protein uptake would be the prelude to cell death.

Relationship between cell kinetic changes and metabolic events during cell senescence in vitro.

Cell kinetic studies performed throughout the lifespan of fibroblasts with a limited lifespan in vitro have led to the conclusion that although division slows down, almost all cells are able to divide until the last subcultivation. The prolongation of the division cycle is primarily due to the impariment of mechanisms preceding DNA synthesis and mitosis. An attempt was made to distinguish between primary and secondary changes and to correlate the findings concerning cell kinetics with alterations observed at the molecular level. A decline in protein synthesis was the first modification detected. The two parameters that are always present during cell senescence in vitro, i.e., and increase in cell volume and a decrease in saturation density could be due respectively to a change in cell permeability and a decline in ribosome synthesis. The latter could also be the step responsible for the limited potential of division.