Adaptive behavior in autism: Minimal clinically important differences on the Vineland-II.

Autism Spectrum Disorder (ASD) is associated with persistent impairments in adaptive abilities across multiple domains. These social, personal, and communicative impairments become increasingly pronounced with development, and are present regardless of IQ. The Vineland Adaptive Behavior Scales, Second Edition (Vineland-II) is the most commonly used instrument for quantifying these impairments, but minimal clinically important differences (MCIDs) on Vineland-II scores have not been rigorously established in ASD. We pooled data from several consortia/registries (EU-AIMS LEAP study, ABIDE-I, ABIDE-II, INFOR, Simons Simplex Collection and Autism Treatment Network [ATN]) and clinical investigations and trials (Stanford, Yale, Roche) resulting in a data set of over 9,000 individuals with ASD. Two approaches were used to estimate MCIDs: distribution-based methods and anchor-based methods. Distribution-based MCID [d-MCID] estimates included the standard error of the measurement, as well as one-fifth and one-half of the covariate-adjusted standard deviation (both cross-sectionally and longitudinally). Anchor-based MCID [a-MCID] estimates include the slope of linear regression of clinician ratings of severity on the Vineland-II score, the slope of linear regression of clinician ratings of longitudinal improvement category on Vineland-II change, the Vineland-II change score maximally differentiating clinical impressions of minimal versus no improvement, and equipercentile equating. Across strata, the Vineland-II Adaptive Behavior Composite standardized score MCID estimates range from 2.01 to 3.2 for distribution-based methods, and from 2.42 to 3.75 for sample-size-weighted anchor-based methods. Lower Vineland-II standardized score MCID estimates were observed for younger and more cognitively impaired populations. These MCID estimates enable users of Vineland-II to assess both the statistical and clinical significance of any observed change. Autism Res 2018, 11: 270-283. © 2017 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: The Vineland Adaptive Behavior Scales (2nd edition; Vineland-II) is the most widely used scale for assessing day-to-day "adaptive" skills. Yet, it is unknown how much Vineland-II scores must change for those changes to be regarded as clinically significant. We pooled data from over 9,000 individuals with ASD to show that changes of 2-3.75 points on the Vineland-II Composite score represent the "minimal clinically-important difference." These estimates will help evaluate the benefits of potential new treatments for ASD.

In silico identification and in vivo validation of miR-495 as a novel regulator of motivation for cocaine that targets multiple addiction-related networks in the nucleus accumbens.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression and are implicated in the etiology of several neuropsychiatric disorders, including substance use disorders (SUDs). Using in silico genome-wide sequence analyses, we identified miR-495 as a miRNA whose predicted targets are significantly enriched in the Knowledgebase for Addiction Related Genes (ARG) database (KARG; http://karg.cbi.pku.edu.cn). This small non-coding RNA is also highly expressed within the nucleus accumbens (NAc), a pivotal brain region underlying reward and motivation. Using luciferase reporter assays, we found that miR-495 directly targeted the 3'UTRs of Bdnf, Camk2a and Arc. Furthermore, we measured miR-495 expression in response to acute cocaine in mice and found that it is downregulated rapidly and selectively in the NAc, along with concomitant increases in ARG expression. Lentiviral-mediated miR-495 overexpression in the NAc shell (NAcsh) not only reversed these cocaine-induced effects but also downregulated multiple ARG mRNAs in specific SUD-related biological pathways, including those that regulate synaptic plasticity. miR-495 expression was also downregulated in the NAcsh of rats following cocaine self-administration. Most importantly, we found that NAcsh miR-495 overexpression suppressed the motivation to self-administer and seek cocaine across progressive ratio, extinction and reinstatement testing, but had no effect on food reinforcement, suggesting that miR-495 selectively affects addiction-related behaviors. Overall, our in silico search for post-transcriptional regulators identified miR-495 as a novel regulator of multiple ARGs that have a role in modulating motivation for cocaine.

Neuronal RNA-binding protein HuD regulates addiction-related gene expression and behavior.

The neuronal RNA-binding protein HuD is involved in synaptic plasticity and learning and memory mechanisms. These effects are thought to be due to HuD-mediated stabilization and translation of target mRNAs associated with plasticity. To investigate the potential role of HuD in drug addiction, we first used bioinformatics prediction algorithms together with microarray analyses to search for specific genes and functional networks upregulated within the forebrain of HuD overexpressing mice (HuDOE ). When this set was further limited to genes in the knowledgebase of addiction-related genes database (KARG) that contains predicted HuD-binding sites in their 3' untranslated regions (3'UTRs), we found that HuD regulates networks that have been associated with addiction-like behavior. These genes included Bdnf and Camk2a, 2 previously validated HuD targets. Since addiction is hypothesized to be a disorder stemming from altered gene expression causing aberrant plasticity, we sought to test the role of HuD in cocaine conditioned placed preference (CPP), a model of addiction-related behaviors. HuD mRNA and protein were upregulated by CPP within the nucleus accumbens of wild-type C57BL/6J mice. These changes were associated with increased expression of Bdnf and Camk2a mRNA and protein. To test this further, we trained HuDOE and wild-type mice in CPP and found that HuDOE mice showed increased cocaine CPP compared with controls. This was also associated with elevated expression of HuD target mRNAs and proteins, CaMKIIα and BDNF. These findings suggest HuD involvement in addiction-related behaviors such as cocaine conditioning and seeking, through increased plasticity-related gene expression.

Social cognition in autism is associated with the neurodevelopment of the posterior superior temporal sulcus.

OBJECTIVE: The posterior superior temporal sulcus (pSTS) plays a critical role in the 'social brain'. Its neurodevelopment and relationship with the social impairment in autism spectrum disorders (ASD) are not well understood. We explored the relationship between social cognition and the neurodevelopment of the pSTS in ASD. METHOD: We included 44 adults with high-functioning ASD and 36 controls. We assessed their performances on the 'Reading the mind in the eyes' test (for 34 of 44 subjects with ASD and 30 of 36 controls), their fixation time on the eyes with eye tracking (for 35 of 44 subjects with ASD and 30 of 36 controls) and the morphology of the caudal branches of the pSTS (length and depth), markers of the neurodevelopment, with structural MRI. RESULTS: The right anterior caudal ramus of the pSTS was significantly longer in patients with ASD compared with controls (52.6 mm vs. 38.3 mm; P = 1.4 × 10-3 ; Cohen's d = 0.76). Its length negatively correlated with fixation time on the eyes (P = 0.03) in the ASD group and with the 'Reading the mind in the eyes' test scores in both groups (P = 0.03). CONCLUSION: Our findings suggest that the neurodevelopment of the pSTS is related to the ASD social impairments.

Increased expression and localization of the RNA-binding protein HuD and GAP-43 mRNA to cytoplasmic granules in DRG neurons during nerve regeneration.

The neuronal-specific RNA-binding protein, HuD, binds to a U-rich regulatory element of the 3' untranslated region (3' UTR) of the GAP-43 mRNA and delays the onset of its degradation. We have recently shown that overexpression of HuD in embryonic rat cortical cells accelerated the time course of normal neurite outgrowth and resulted in a twofold increase in GAP-43 mRNA levels. Given this evidence, we sought to investigate the involvement of HuD during nerve regeneration. It is known that HuD protein and GAP-43 mRNA are expressed in the dorsal root ganglia (DRG) of adult rat and that GAP-43 is upregulated in DRG neurons during regeneration. In this study, we examined the expression patterns and levels of HuD and GAP-43 mRNA in DRG neurons following sciatic nerve injury using a combination of in situ hybridization, immunocytochemistry, and quantitative RT-PCR. GAP-43 and HuD expression increased in the ipsilateral DRG during the first 3 weeks of regeneration, with peak values seen at 7 days postcrush. At this time point, the levels of HuD and GAP-43 mRNAs in the ipsilateral DRG increased by twofold and sixfold, respectively, relative to the contralateral DRG. Not only were the temporal patterns of expression of HuD protein and GAP-43 mRNA similar, but also they were found to colocalize in the cytoplasm of DRG neurons. Moreover, both molecules were distributed in cytoplasmic granules containing ribosomal RNA. In conclusion, our results suggest that HuD is involved in the upregulation of GAP-43 expression observed at early stages of peripheral nerve regeneration.

Towards global and long-term neurological gene therapy: unexpected transgene dependent, high-level, and widespread distribution of HSV-1 thymidine kinase throughout the CNS.

One of the challenges of neurological gene therapy for the treatment of chronic neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases, is achieving high levels, widespread distribution, and long-lived transgene expression in the brain. Here, following the intracerebral injection of a recombinant adenovirus (RAd) encoding herpes simplex virus type 1 thymidine kinase (HSV1-TK), we detect very high levels of HSV1-TK immunoreactivity throughout the brain both ipsilaterally and contralaterally to the injection site, for up to 12 months following vector administration. This study concludes that long-term, high-level, and anatomically distributed HSV1-TK immunoreactivity can be obtained, and that this is most likely due to transgene-specific properties, because neither the distribution nor the longevity were observed for the transgene beta-galactosidase encoded by a co-injected vector. Thus, we demonstrate that transgene expression can be achieved over widespread areas of the rodent brain, even 12 months after a single injection of first-generation adenovirus vector.

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: impact on hormone secretion.

OBJECTIVE: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/beta-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli beta-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). DESIGN: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/beta-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. METHODS: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for beta-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. RESULTS: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/beta-gal showed widespread expression of the beta-galactosidase transgene around the injection areas. CONCLUSIONS: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.

Thyroid-stimulating hormone and growth hormone release alterations induced by mosquito larvae proteins on pituitary cells.

Mosquito larvae crude extract has shown to modulate cell proliferation of different mouse epithelial as well as human mononuclear cell populations in vivo and in vitro. A soluble fraction of the extract, with a molecular weight ranging from 12 to 80 kD, also showed an inhibitory effect on the proliferation of mouse hepatocytes. This effect disappeared after heating the extract at 90 degrees C for 60 min, suggesting that some proteinaceous molecule is involved. We report the effect of dialysed extract (MW >12 kD) on the concentration of both thyroid-stimulating hormone (TSH) and growth hormone (GH) in an incubation medium of pituitary cells from normal and oestrogenised rats. Time- and dose-dependent response of both hormones resulted in increasing TSH levels. Concentrations of GH were lower in the treated than in control pituitary cells. The time elapsed until the finding of differences suggests the presence in the mosquito extract of some protein binding the hormone. The differences were not due to lethal toxic effects since the Trypan blue viability test showed no differences between control and treated cells. Furthermore, the effect disappeared when the extract had previously been heated at 90 degrees C for 60 min. Finally, our results suggest the presence of some proteins in the mosquito Culex pipiens L. larvae, which would act as a pituitary hormone regulator.

Neuroendocrinology of aging: the potential of gene therapy as an interventive strategy.

OBJECTIVE: This paper reviews the current status of gene therapy in the neuroendocrine system and discusses the interventive potential of this methodology for neuroendocrine pathologies associated with aging. BACKGROUND AND RESULTS: A brief description is first presented of the viral-vector-based gene delivery systems being currently used in the neuroendocrine system, namely the adenoviral and herpetic (HSV1) vector systems. Next, an account of the neuroendocrine pathologies for which gene therapy approaches in animal models are being implemented is provided. This includes the treatment of experimental pituitary tumors by adenoviral-vector-mediated transfer of the suicide gene for the HSV-1 thymidine kinase. At the hypothalamic level, an adenovirus harboring the cDNA for arginine vasopressin has been used in Brattleboro rats to correct their diabetes insipidus. Next, the interventive potential of gene therapy for correcting age-associated neurodegenerative processes at neuroendocrine level is outlined. Finally, the role that emerging technologies may play in the development of future genetic therapies for aging is considered. CONCLUSION: Although effective implementation of gene therapy strategies still faces significant technical obstacles, these are likely to be progressively overcome as gene delivery systems are refined.

Twenty-four-hour profiles of serum prolactin and luteinizing hormone in anoestrous crossbred bitches.

The dynamics of prolactin (PRL) and luteinizing hormone (LH) secretion in the anoestrous bitch is poorly known. Therefore, the objective of this study was to characterize the 24 h profiles of serum PRL and LH in crossbred anoestrous bitches and to assess whether a relationship exists between the secretory patterns of these two hormones. Serum PRL and LH concentrations were measured in 10 healthy anoestrous crossbred bitches at 145 min intervals for 24 h. During the experiment the animals received continuous artificial illumination and remained undisturbed except at the time of blood sampling. Serum PRL was measured by a homologous enzyme-linked immunosorbent assay, whereas LH and progesterone (P4) were determined by radioimmunoassay. The anoestrous state of the bitches was assessed by vaginal cytology, vaginoscopy and physical examination. Two groups of animals were identified according to their PRL levels: a high PRL group (n = 3, mean +/- SEM 12.3 +/- 2.7 ng/ml) and a low PRL group (n = 7, mean +/- SEM: 2.5 +/- 0.9 ng/ml). In the low PRL group, the PRL profiles were flat and did not show any significant circadian pattern. Nevertheless, occasional single-point peaks were detected in some of the bitches. In the high PRL group the individual PRL profiles were variable. To detect the presence of a circadian change of PRL concentrations, two different sets of time windows (TW) of sampling were studied. The first set was: day [TW1A, samples 1-5 (0900-1840 h)] and night [TW1B, samples 6-10 (2105-0645 h)]. The second set was chosen after visual inspection of the average PRL profiles for both (high and low) groups: [TW2A, samples 3-7 (1350-2330 h) and TW2B, samples 1-2 and 8-10 (0155-1125 h)]. PRL concentrations were not significantly different between day and night. In the high PRL group, but not in the low PRL group, average serum PRL was significantly (p < 0.01) higher in TW2A than TW2B. In both groups serum LH levels were more homogeneous than PRL levels. Neither TW showed circadian changes in LH patterns of secretion (TW1A versus TW1B, p < 0.69; TW2A versus TW2B, p < 0.88). On the other hand, bitches in the high PRL group showed significantly (p < 0.01) lower serum LH levels than those in the low PRL group of animals. Serum PRL concentrations presented a significant inverse correlation with LH concentrations (r=-0.21, p < 0.03) and a significant positive correlation with P4 concentrations across the study (0.92, p < 0.01). It is concluded that in anoestrous crossbred bitches serum PRL is highly variable and inversely related to LH. No circadian rhythm of PRL secretion appears to exist in most anoestrous bitches.

Gene therapy in the neuroendocrine system: its implementation in experimental models using viral vectors.

Gene therapy, the transfer of genetic material for therapeutic purposes, has undergone an explosive development in the last few years. Within this context, development of gene therapy approaches for the neuroendocrine system, while incipient, has already generated a core of results which emerge as a promising area of research in neuroendocrinology. The present review presents a brief description of the viral vector-based gene delivery systems being currently used in neuroendocrinology, namely the adenoviral and herpes simplex type-1 (HSV-1)-derived vector systems, as well as an updated account of neuroendocrine pathologies for which gene therapy approaches in animal models are being implemented is provided. Current research efforts include treatment of experimental pituitary tumors by adenoviral vector-mediated transfer of the suicide gene for the HSV-1 thymidine kinase, which converts the prodrug ganciclovir into a toxic metabolite. An adenoviral vector encoding the human retinoblastoma suppressor oncogene has also been successfully used to rescue the phenotype of spontaneous pituitary tumors of the pars intermedia in mice. At the hypothalamic level, an adenovirus harboring the cDNA for arginine vasopressin has been used in Brattleboro rats to correct diabetes insipidus for several weeks. The last part of the review outlines the potential of gene therapy to correct age-associated neurodegenerative processes at the neuroendocrine level. Although effective implementation of gene therapy strategies still faces significant technical obstacles, these are likely to be progressively overcome as gene delivery systems are being improved.

Mosquito larvae proteins modulate luteinizing hormone and prolactin release in pituitary cells of normal and estrogenized rats.

Whilst looking for vertebrate growth factor homologues in insects, we found that a soluble fraction of a 12-80 kDa molecular weight band peaking at 25 kDa, isolated from mosquito larvae extracts by gel permeation chromatography, had a modulatory effect on mouse hepatocytes and adult human mononuclear cell proliferation. The effect disappeared after heating the extract at 90 degrees C for 30 min, suggesting that the active factor may be a protein. In order to determine the activity of the extract on cell function, we assessed the effect of the extract on pituitary hormone secretion in vitro. We assayed a dialyzed fraction (MW greater than 12 kDa) of mosquito larvae for its effect on the release of luteinizing hormone (LH) and prolactin (PRL) from dispersed rat pituitary cells. In normal anterior pituitary (AP) cells we found that the extract had a stimulatory effect on LH release but an inhibitory action on prolactin secretion. In AP cells obtained from estrogen-induced hyperplasia, the extract had an inhibitory effect on prolactin secretion. In all cases the effects were time- and dose-dependent. Interference of the mosquito proteins with the radioimmunoassay was checked and found to be negligible. After a 60 min incubation, cell viability was comparable in control and treated cells. Furthermore, the biological effect of the extract was thermally unstable. Our results suggest that mosquito larvae may share common factors with mammals, probably peptidic in nature, which are able to modulate cell function.

Effect of lactobacilli, bifidobacteria and inulin on the formation of aberrant crypt foci in rats.

BACKGROUND: Our studies were aimed at investigating the effect of lactic acid producing bacteria (LAB) or inulin, a natural source of non-digestible oligosaccharides derived from chicory, on the induction by carcinogens of aberrant crypt foci (ACF) in the colon, which are considered to be early precursor lesions of neoplasia. METHODS: Strains of Bifidobacterium longum, Lactobacillus casei and Lactobacillus acidophilus were administered to rats fed a purified high starch diet, under a variety of treatment protocols including daily gavage, via the drinking water and in the diet. The rats were treated with methyl-N-nitrosourea, 1,2-dimethylhydrazine, or azoxymethane (AOM) to induce ACF. RESULTS: In general, no consistent significant changes in ACF numbers were detected in these experiments. In one study, the basal diet of the rats was changed to one containing a higher level of fat (corn oil). Under these conditions, a significant decrease in AOM-induced colonic ACF was seen in rats given L. acidophilus or inulin. In a concurrent group of animals fed a low fat diet, no significant decrease in ACF was observed. CONCLUSIONS: The results indicate that the type of diet fed can influence the detection of protective effects of LAB and oligosaccharides and that against the background of a diet with a level of fat typical of a Western diet, evidence for a protective effect of L. acidophilus and inulin towards colon cancer was obtained

Adenovirus-mediated herpes simplex virus type-1 thymidine kinase gene therapy suppresses oestrogen-induced pituitary prolactinomas.

We tested the hypothesis that gene transfer using recombinant adenovirus vectors (RAds) expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) might offer an alternative therapeutic approach for the treatment of pituitary prolactinomas that do not respond to classical treatment strategies. HSV1-TK converts the prodrug ganciclovir (GCV) to GCV monophosphate, which is in turn further phosphorylated by cellular kinases to GCV triphosphate, which is toxic to proliferating cells. One attractive feature of this system is the bystander effect, whereby untransduced cells are also killed. Our results show that RAd/HSV1-TK in the presence of GCV is nontoxic for the normal anterior pituitary (AP) gland in vitro, but causes cell death in the pituitary tumor cell lines GH3, a PRL/GH-secreting cell line, and AtT20, a corticotrophic cell line. We have used sulpiride- and oestrogen-induced lactotroph hyperplasia within the rat AP gland as an in vivo animal model. Intrapituitary infection of rats bearing oestrogen-induced lactotroph hyperplasia, with RAd/ HSV1-TK and subsequent treatment with GCV, decreases plasma PRL levels and reduces the mass of the pituitary gland. More so, there were no deleterious effects on circulating levels of other AP hormones, suggesting that the treatment was nontoxic to the AP gland in situ. In summary, our results show that suicide gene therapy using the HSV1-TK transgene could be further developed as a useful treatment to complement current therapies for prolactinomas.

Viral vectors for gene delivery and gene therapy within the endocrine system.

The transfer of genetic material into endocrine cells and tissues, both in vitro and in vivo, has been identified as critical for the study of endocrine mechanisms and the future treatment of endocrine disorders. Classical methods of gene transfer, such as transfection, are inefficient and limited mainly to delivery into actively proliferating cells in vitro. The development of viral vector gene delivery systems is beginning to circumvent these initial setbacks. Several kinds of viruses, including retrovirus, adenovirus, adeno-associated virus, and herpes simplex virus, have been manipulated for use in gene transfer and gene therapy applications. As different viral vector systems have their own unique advantages and disadvantages, they each have applications for which they are best suited. This review will discuss viral vector systems that have been used for gene transfer into the endocrine system, and recent developments in viral vector technology that may improve their use for endocrine applications - chimeric vectors, viral vector targeting and transcriptional regulation of transgene expression.

Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: implications for clinical trials.

The long-term consequences of adenovirus-mediated conditional cytotoxic gene therapy for gliomas remain uncharacterized. We report here detection of active brain inflammation 3 months after successful inhibition of syngeneic glioma growth. The inflammatory infiltrate consisted of activated macrophages/microglia and astrocytes, and T lymphocytes positive for leucosyalin, CD3 and CD8, and included secondary demyelination. We detected strong widespread herpes simplex virus 1 thymidine kinase immunoreactivity and vector genomes throughout large areas of the brain. Thus, patient evaluation and the design of clinical trials in ongoing and future gene therapy for brain glioblastoma must address not only tumor-killing efficiency, but also long-term active brain inflammation, loss of myelin fibers and persistent transgene expression.

Homeostasis, thymic hormones and aging.

The thymic-pituitary axis constitutes a bidirectional circuit where the ascending feedback loop is effected by thymic factors of epithelial origin. The aim of the present article is, first, to introduce the idea of an immune-neuroendocrine homeostatic network in higher animals. Next, the relevance of the thymus in this network and the possible role of this gland in the neuroendocrine imbalances associated with aging are discussed. A number of studies are next reviewed which show that the endocrine thymus produces several bioactive molecules, generally called thymic hormones, which in addition to possessing immunoregulatory properties are also active on nervous and endocrine circuits. In particular, the reported activities of thymosin fraction five, thymosin alpha 1 and thymosin beta 4 on beta-endorphin, adrenocorticotropic hormone, glucocorticoids, luteinizing hormone-releasing hormone and luteinizing hormone secretion in different animal and cell models are reviewed. The known hypophysiotropic actions of other thymic hormones like thymulin, homeostatic thymus hormone and thymus factor are also summarized, and the impact of aging on pituitary responsiveness to thymic hormones is discussed. As a conclusion, it is proposed that in addition to its central role in the regulation of the immune function, the thymus gland may extend its influence to nonimmunologic components of the body, including the neuroendocrine system. The early onset of thymus involution might, therefore, act as a triggering event which would initiate the gradual decline in homeostatic potential that characterizes the aging process.

The thymus-pituitary axis and its changes during aging.

The pituitary-thymic axis constitutes a bidirectional circuit where the ascending feedback loop is effected by thymic factors of epithelial origin. The aim of the present article is to review the evidence demonstrating that aging brings about a progressive disruption in the integration of this network. In doing so, we briefly review the experimental evidence supporting the view that immune and neuroendocrine aging are interdependent processes. The advantages and limits of the nude mouse as a model of thymus-dependent accelerated aging is also discussed. Next, we review a number of studies which show that the endocrine thymus produces several bioactive molecules, generally called thymic hormones, which in addition to possessing immunoregulatory properties are also active on nervous and endocrine circuits. In particular, the reported activities of thymosin fraction 5 (TF5), thymosin alpha-1 and thymosin beta-4 on beta-endorphin, ACTH, glucocorticoids, LHRH and LH secretion in different animal and cell models are reviewed. The known hypophysiotropic actions of other thymic hormones like thymulin, homeostatic thymus hormone (HTH) and thymus factor are summarized. Aging has a significant impact on pituitary responsiveness to thymic hormones. Thus, it has been reported that TF5 and HTH have thyrotropin-inhibiting activity in young but not in old rats. Furthermore, intravenous administration of HTH was also able to reduce plasma GH and increase corticosterone levels in both young and old rats, although these responses were much weaker in the old animals. Further evidence on this topic is discussed. It is proposed that in addition to its central role in the regulation of the immune function, the thymus gland may extend its influence to nonimmunologic components of the body, including the neuroendocrine system. The early onset of thymus involution might therefore act as a triggering event which would initiate the gradual decline in homeostatic potential that characterizes the aging process.

Thymulin stimulates prolactin and thyrotropin release in an age-related manner.

Thymulin is a Zn-bound nonapeptide produced by the thymic epithelial cells (TEC) whose secretion is modulated by prolactin (PRL) and thyroid hormones, among other hormones. We assessed the ability of thymulin to influence the release of PRL and thyroid stimulating hormone (TSH) from dispersed anterior pituitary (AP) cells from young, middle-aged and senescent Sprague-Dawley female rats. Perifused and incubated AP cells were used in different sets of experiments and PRL and TSH release was measured by radioimmunoassay. Perifusion of young and senescent AP cells with thymulin doses ranging from 10(-8) to 10(-5) M gave a logarithmic dose response pattern for both hormones. Supernatants from TEC lines also showed PRL and TSH secretagogue activity. Hormone release was always lower in the senescent cells. AP cells incubated with 10(-8) to 10(-3) M thymulin showed a time- and dose-dependent response for both hormones, the latter being bell-shaped with a maximum at 10(-7) M thymulin. Preincubation of thymulin with an anti-thymulin serum completely quenched the secretagogue activity of the thymic hormone. Coincubation of thymulin with TRH revealed a synergistic release of PRL and TSH in AP cells from all age groups. The calcium chelator EGTA but not the calcium ionophore A23187, blocked the hormone-releasing activity of thymulin in AP cells. The cAMP enhancers, caffeine, NaF and forskolin, significantly increased the thymulin-stimulated release of PRL and TSH, while trifluoperazine, a protein kinase C inhibitor, had no effect. The inositol phosphate enhancer LiCl, potentiated the action of thymulin on PRL and TSH. The present results suggest that the TRH-like activity documented here for thymulin is a receptor-mediated effect which appears to involve calcium, cAMP and inositol phosphates. Senescence but not middle age, appears to impair AP cell responsiveness to thymulin.

Influence of carcinogen binding by lactic acid-producing bacteria on tissue distribution and in vivo mutagenicity of dietary carcinogens.

The aims of this investigation were to determine whether viable cultures of lactic acid-producing organisms (LAB) can bind dietary carcinogens and to assess the consequences of binding for the absorption from the gut, distribution in the body and in vivo genotoxicity of ingested carcinogens. The carcinogens used in this study were ones known to be present in the human diet, namely benzo[a]pyrene (B(a)P, aflatoxin B1 (AFB1) and the cooked food carcinogens 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 5-phenyl-2-amino-1-methylimidazo [4,5-f]pyridine (PhIP) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). They represent a range of structural types so that the specificity of any binding effects could be addressed. Of the carcinogens tested, B(a)P and Trp-P-2 were bound most effectively by the two LAB strains Bifidobacterium longum and Lactobacillus acidophilus. AFB1 was poorly bound, while MeIQx, MeIQ, PhIP and IQ were bound to an intermediate degree. The extent of the binding of the heterocyclic amine carcinogens was dependent on the pH conditions during incubation and this effect was more apparent with B. longum than with L. acidophilus. Using the host-mediated assay (HMA), an in vivo bacterial mutation assay, it was demonstrated that the administration of bacterial cell suspensions of B. longum and L. acidophilus did not lead to a reduction in induced mutagenicity by MeIQ, MeIQx or Trp-P-2, detectable in the liver of treated mice compared with controls. The lack of a protective effect could not be attributed to a short period of contact between bacterial cells and mutagens, since similar results were obtained after preincubating bacteria and mutagens together at pH 5 for 50-60 min, to maximize the binding, before gavaging the mice. Lack of activity of B(a)P in the HMA prevented the determination of the effect of LAB on genotoxicity of the polycyclic aromatic hydrocarbon. However, it is clear from the radiolabel distribution study that the amount of the carcinogen entering the blood was not significantly reduced by B. longum administration. In addition, the amount of radiolabelled B(a)P that reached the target organs (liver, lungs and heart) was also not affected by the LAB administration. A similar lack of inhibitory effect of B. longum on blood concentration and accumulation in the liver of Trp-P-2 was apparent. The results of the present study suggest that although LAB may bind carcinogens in vitro, this does not lead to major changes in absorption and distribution of carcinogens in the body, or in their genotoxic activity in the liver.