Curcumin ameliorates autoimmune diabetes. Evidence in accelerated murine models of type 1 diabetes.

Type 1 diabetes (T1DM) is a T cell-mediated autoimmune disease that selectively destroys pancreatic ß cells. The only possible cure for T1DM is to control autoimmunity against ß cell-specific antigens. We explored whether the natural compound curcumin, with anti-oxidant and anti-inflammatory activities, might down-regulate the T cell response that destroys pancreatic ß cells to improve disease outcome in autoimmune diabetes. We employed two accelerated autoimmune diabetes models: (i) cyclophosphamide (CYP) administration to non-obese diabetic (NOD) mice and (ii) adoptive transfer of diabetogenic splenocytes into NODscid mice. Curcumin treatment led to significant delay of disease onset, and in some instances prevented autoimmune diabetes by inhibiting pancreatic leucocyte infiltration and preserving insulin-expressing cells. To investigate the mechanisms of protection we studied the effect of curcumin on key immune cell populations involved in the pathogenesis of the disease. Curcumin modulates the T lymphocyte response impairing proliferation and interferon (IFN)-γ production through modulation of T-box expressed in T cells (T-bet), a key transcription factor for proinflammatory T helper type 1 (Th1) lymphocyte differentiation, both at the transcriptional and translational levels. Also, curcumin reduces nuclear factor (NF)-κB activation in T cell receptor (TCR)-stimulated NOD lymphocytes. In addition, curcumin impairs the T cell stimulatory function of dendritic cells with reduced secretion of proinflammatory cytokines and nitric oxide (NO) and low surface expression of co-stimulatory molecules, leading to an overall diminished antigen-presenting cell activity. These in-vitro effects correlated with ex-vivo analysis of cells obtained from curcumin-treated mice during the course of autoimmune diabetes. These findings reveal an effective therapeutic effect of curcumin in autoimmune diabetes by its actions on key immune cells responsible for ß cell death.

Cell-based interventions to halt autoimmunity in type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) results from death of insulin-secreting ß cells mediated by self-immune cells, and the consequent inability of the body to maintain insulin levels for appropriate glucose homeostasis. Probably initiated by environmental factors, this disease takes place in genetically predisposed individuals. Given the autoimmune nature of T1DM, therapeutics targeting immune cells involved in disease progress have been explored over the last decade. Several high-cost trials have been attempted to prevent and/or reverse T1DM. Although a definitive solution to cure T1DM is not yet available, a large amount of information about its nature and development has contributed greatly to both the improvement of patient's health care and design of new treatments. In this study, we discuss the role of different types of immune cells involved in T1DM pathogenesis and their therapeutic potential as targets and/or modified tools to treat patients. Recently, encouraging results and new approaches to sustain remnant ß cell mass and to increase ß cell proliferation by different cell-based means have emerged. Results coming from ongoing clinical trials employing cell therapy designed to arrest T1DM will probably proliferate in the next few years. Strategies under consideration include infusion of several types of stem cells, dendritic cells and regulatory T cells, either manipulated genetically ex vivo or non-manipulated. Their use in combination approaches is another therapeutic alternative. Cell-based interventions, without undesirable side effects, directed to block the uncontrollable autoimmune response may become a clinical reality in the next few years for the treatment of patients with T1DM.

Curcumin acts anti-proliferative and pro-apoptotic in human meningiomas.

Meningiomas, the most frequent benign intracranial and intraspinal types of tumors are normally removed by surgery. Complications can occur when the tumor is critically localized and cannot be completely removed or when comorbidities of the mostly elder patients increase the general surgical risk. Thus, alternate medical treatment concepts for the therapy of meningiomas would be desirable. Curcumin, the active ingredient of the spice plant Curcuma longa has shown anti-tumorigenic actions in many different types of tumors and therefore, its effect on growth and apoptosis of meningioma cells was studied in the present paper. In vitro, treatment of the human Ben-Men-1 meningioma cell line and of a series of 21 primary human meningioma cell cultures with curcumin (1-20 µM) strongly reduced the proliferation in all cases in a dose dependent manner. Cell cycle analysis by fluorescence-activated cell sorting showed growth arrest at G2/M phase, which was confirmed by demonstrating the corresponding modulation of proteins involved in G2/M arrest by immunoblotting and/or confocal laser microscopy. High dosages (20, 50 µM) of curcumin induced a significant increase of apoptosis in Ben-Men-1 and primary meningioma cell cultures as demonstrated by morphological changes of cell nuclei, DNA fragmentation, translocation of cell membrane associated phosphatidyl serine and the induction of apoptotic-acting cleaved caspase-3. Our results suggest that the multi-targeting drug curcumin has potent anti-tumorigenic actions in meningioma cells and might therefore be a putative candidate for the pharmacological treatment of meningiomas.

Cotransplantation of marginal mass allogeneic islets with 3D culture-derived adult human skin cells improves glycemia in diabetic mice.

Islet transplantation represents a therapeutic option for type 1 diabetes (T1D). Long-term viability of transplanted islets requires improvement. Mesenchymal stromal cells (MSCs) have been proposed as adjuvants for islet transplantation facilitating grafting and functionality. Stem cell aggregation provides physiological interactions between cells and enhances the in situ concentration of modulators of inflammation and immunity. We established a hanging-drop culture of adult human skin fibroblast-like cells as spheroids, and skin spheroid-derived cells (SphCs) were characterized. We assessed the potential of SphCs in improving islet functionality by cotransplantation with a marginal mass of allogeneic islets in an experimental diabetic mouse model and characterized the secretome of SphCs by mass spectrometry-based proteomics. SphCs were characterized as multipotent progenitors and their coculture with anti-CD3 stimulated mouse splenocytes decreased CD4+ T cell proliferation with skewed cytokine secretion through an increase in the Th2/Th1 ratio profile. SphCs-conditioned media attenuated apoptosis of islets induced by cytokine challenge in vitro and importantly, intratesticular SphCs administration did not show tumorigenicity in immune-deficient mice. Moreover, SphCs improved glycemic control when cotransplanted with a marginal mass of allogeneic islets in a diabetic mouse model without pharmacological immunosuppression. SphCs' protein secretome differed from its paired skin fibroblast-like counterpart in containing 70% of up- and downregulated proteins and biological processes that overall positively influenced islets such as cytoprotection, cellular stress, metabolism, and survival. In summary, SphCs improved the performance of transplanted allogeneic islets in an experimental T1D model, without pharmacological immunosuppression. Future research is warranted to identify SphCs-secreted factors responsible for islets' endurance.

Novel medical therapies for pituitary tumors.

Despite considerable progress, there is still no medical treatment available for some kinds of pituitary tumors, in particular hormone inactive adenomas and corticotroph pituitary tumors. Surgical removal or at least debulking of the tumor is the only option to treat these kinds of tumors apart from rarely applied radiotherapy. Moreover, treatment resistance is present in a considerable proportion of patients bearing pituitary tumors, for which medical treatment regimens are already available (prolactinomas, somatotroph adenomas). Thus, novel or improved medical treatment strategies would be desirable. Here, we summarize preclinical and clinical findings about the hormone and growth-suppressive action of various drugs, which will probably lead to novel future medical treatment concepts for pituitary tumors.

Vigilance on use of drugs, herbal products, and food supplements during pregnancy: focus on fosfomycin.

PURPOSE: Urinary tract infection (UTI) is defined as a common bacterial infection that can lead to significant morbidity such as stricture, fistula, abscess formation, bacteremia, sepsis, pyelonephritis, and kidney dysfunction with a mortality rates reported of 1% in men and 3% in women because of development of pyelonephritis. UTIs are more common in women and the 33% of them require antimicrobials treatment for at least one episode by the age of 24 years. UTIs are the most common infections observed during pregnancy and up to 30% of mothers with not treated asymptomatic bacteriuria may develop acute pyelonephritis which consequently can be associated to adverse maternal and fetal outcomes. All bacteriuria in pregnancy should be treated with antimicrobial treatments being safe for both the mother and the fetus. Approximately one every four women receives prescription of antibiotic treatment during pregnancy, nearly 80% of all the prescription medications during gestation. The use of fosfomycin to treat cystitis in pregnancy generally considered safe and effective. Even though use on antibiotics for urinary tract infections is considered generally safe for the fetus and mothers, this opinion is not based on specific studies monitoring the relationship of among urinary infections, consumption of antibiotics, and pregnancy outcomes. MATERIALS AND METHODS: On this basis we decided to analyze data from the database of our multicenter study PHYTOVIGGEST, reporting data from 5362 pregnancies, focusing on use of fosfomycin. Principal outcomes of pregnancy in women treated with fosfomycin were taken into consideration. RESULTS: Women who have been treated with urinary antibiotics during the pregnancy were 183. With respect to the total number of pregnancies of our sample, these women represented the percentage of 3.49% (187/5362). Analysis of different outcomes of pregnancy such as gestational age, neonatal weight, and neonatal Apgar index did not show any significant difference. At the same time, analysis of data of pregnancy complicancies (such as urgent cesarean delivery, use of general anesthesia, need to induce labor) did not show any difference in women taking fosfomycin during pregnancy and those not taking it. CONCLUSIONS: Our data, based on a large number of pregnancies, confirm the safety use of fosfomycin use in pregnancy.

Cotransplantation of marginal mass allogeneic islets with 3D culture-derived adult human skin cells improves glycemia in diabetic mice

Islet transplantation represents a therapeutic option for type 1 diabetes (T1D). Long-term viability of transplanted islets requires improvement. Mesenchymal stromal cells (MSCs) have been proposed as adjuvants for islet transplantation facilitating grafting and functionality. Stem cell aggregation provides physiological interactions between cells and enhances the in situ concentration of modulators of inflammation and immunity. We established a hanging-drop culture of adult human skin fibroblast-like cells as spheroids, and skin spheroid-derived cells (SphCs) were characterized. We assessed the potential of SphCs in improving islet functionality by cotransplantation with a marginal mass of allogeneic islets in an experimental diabetic mouse model and characterized the secretome of SphCs by mass spectrometry-based proteomics. SphCs were characterized as multipotent progenitors and their coculture with anti-CD3 stimulated mouse splenocytes decreased CD4+ T cell proliferation with skewed cytokine secretion through an increase in the Th2/Th1 ratio profile. SphCs-conditioned media attenuated apoptosis of islets induced by cytokine challenge in vitro and importantly, intratesticular SphCs administration did not show tumorigenicity in immune-deficient mice. Moreover, SphCs improved glycemic control when cotransplanted with a marginal mass of allogeneic islets in a diabetic mouse model without pharmacological immunosuppression. SphCs' protein secretome differed from its paired skin fibroblast-like counterpart in containing 70% of up- and downregulated proteins and biological processes that overall positively influenced islets such as cytoprotection, cellular stress, metabolism, and survival. In summary, SphCs improved the performance of transplanted allogeneic islets in an experimental T1D model, without pharmacological immunosuppression. Future research is warranted to identify SphCs-secreted factors responsible for islets' endurance.

Peroperative control of surgical infections.

The overall incidence of post-surgical infection actually amount to 3-10%, in different multicentric trial, although the data may underrepresent the true incidence of such infections owing to increase of day-surgery. Antibiotic prophylaxis rapresents the first choice in the management of surgical patients, which standardization and selection can determine a real protection for all the operating time. Standardization of intraoperative procedure, considering utility of a multistep precautionary measure and the weight of these measures on post-operative stay of patients, may be an arm for control really post-operative infectious complications, according with control of sterilization's procedures and diffusion of dedicated device.

Transcriptional targeting to anterior pituitary lactotrophic cells using recombinant adenovirus vectors in vitro and in vivo in normal and estrogen/sulpiride-induced hyperplastic anterior pituitaries.

The use of pituitary cell type-specific promoters is a powerful molecular tool to achieve pituitary cell type-specific transcriptional targeting of transgenes encoded by viral vectors. It has recently been proposed that transcriptional targeting of therapeutic genes could be harnessed as a gene therapy strategy for the treatment of pituitary disease. We describe the successful use of the human PRL promoter (hPrl) encoded within recombinant adenovirus vectors to target transgene expression of Herpes Simplex Virus Type 1-Thymidine Kinase (HSV1-TK) or beta-galactosidase to lactotrophic cells in vitro and in vivo. Functionally, the restriction of expression of HSV1-TK to lactotrophic tumor cells, using the hPrl promoter, resulted in the cell type-specific induction of apoptosis in the lactotrophic GH3 tumor cell line, in the presence of ganciclovir (GCV). In the corticotrophic AtT20 cell line, we detected neither HSV1-TK expression, nor apoptosis in the presence of GCV. The hPrl promoter encoded within a recombinant adenoviral vector also restricted transgene expression to lactotrophic cells in primary anterior pituitary (AP) cultures, and importantly, within the anterior pituitary gland in vivo. When the HSV1-TK driven by hPrl promoter was used in an in vivo model ofestrogen/sulpiride lactotroph induced hyperplasia within the AP in situ, the treatment was not effective in either reducing the weight of the gland, the number of lactotrophic cells within the transduced area in vivo, or the circulating PRL levels. This is in contrast to the human cytomegalovirus promoter (hCMV) driving expression of HSV1-TK in the same experimental paradigm, which was effective in reducing pituitary weight and circulating PRL levels. Our results have important implications in the design of gene therapy strategies for pituitary tumors. We demonstrate that both the choice of the in vivo animal model, i.e. adenoma in the AP gland in situ, and the particular gene therapy strategy chosen, i.e. use of strong ubiquitous promoters vs. weaker but cell type-specific promoters, determine the experimental therapeutic outcome.

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.

Intracellular retention of the corticotrophin-releasing hormone (CRH) precursor within COS-7 cells.

We investigated the intracellular localization of CRH in transiently transfected COS-7 cells expressing the full-length rat corticotropin-releasing hormone (CRH) precursor cDNA. CRH synthesized by transfected COS-7 cells is mainly stored intracellularly. In contrast, CHO-K1 cells expressing the same CRH precursor stored and released equal amounts of immunoreactive (IR)-CRH. Ultrastructural analysis revealed that CRH is stored in electron-dense aggregates in the RER of transiently transfected COS-7 cells and does not migrate into the Golgi apparatus. On the basis of the different intracellular localization, storage, and release of CRH in COS-7 and CHO-K1 cells, we hypothesize that the intracellular trafficking of CRH within the constitutive secretory pathway for protein secretion not only depends on its primary amino acid sequence but might also be influenced by intracellular conditions or factors.

Procorticotrophin-releasing hormone: endoproteolytic processing and differential release of its derived peptides within AtT20 cells.

Procorticotrophin-releasing hormone (proCRH) is expressed mainly in the hypothalamus and in the placenta, where it undergoes tissue-specific endoproteolysis. Our results show that within stably transfected AtT20/D16V cells proCRH is cleaved to generate two fragments of approximately 8 and 3 kDa which could account for proCRH(125-194) and proCRH(125-151), respectively, and a 4.5 kDa product which could account for mature IR-CRH(1-41). The immunofluorescence staining patterns for IR-CRH and IR-ACTH and their response of secretagogues indicate targeting of proCRH and POMC to the secretory pathway in transfected AtT20 cells. In this work, we have used a unique set of specific RIAs and IRMAs to the full length POMC and proCRH molecules and several products of endoproteolytic processing to assess if they could be released differentially in response to stimulation. Although the release of both IR-ACTH and IR-CRH peptides from transfected AtT20 cells is stimulated in response to exposure to high potassium stimulation (51 mM KCl/SmM CaCl2), the sorting index (SI) suggests that mature ACTH is sorted to the regulated secretory pathway 2.1-fold more efficiently than mature CRH(1-41). Mature ACTH is also sorted to the regulated secretory pathway 9-fold more efficiently than IR-proCRH(125-151). Also, mature CRH(1-41) is sorted to the regulated secretory pathway 3-fold more efficiently than IR-proCRH(125-151). These results therefore indicate that the intracellular mechanisms for the storage and release of POMC, proCRH and their endoproteolytic products differ and would sustain the hypothesis that within mammalian peptidergic cells, different biologically active peptides originating from the same or different precursor molecules, could be differentially released in response to specific stimuli. This would give these cells the capacity to finely regulate neurotransmitter release in response to environmental and physiological demands.

Corticotrophin-releasing hormone receptor type 1: generation and characterization of polyclonal antipeptide antibodies and their localization in pituitary cells and cortical neurones in vitro.

Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays a major role in regulating the endocrine response to stress. CRH acts by first binding to specific receptors on the plasma membrane of target cells. A CRH receptor from a human corticotroph adenoma and rat brain has recently been cloned (CRH-R1). In this paper, we have chosen three different peptide sequences within the CRH-R1 molecule which bear no similarity to other members of this receptor subfamily (or indeed any known protein) and which are likely to be exposed on the surface of the native protein, for antibody production. Some of these fragments produced antipeptide antibodies of good titre which cross-reacted with the CRH-R1 receptor expressed in transiently transfected COS-7 cells and in tissue extracts from rat cerebellum, cortex, pituitary gland and human myometrium, both in Western blots and in liquid-phase radioimmunoassay. We used immunofluorescence techniques to localize the CRH receptor in transiently transfected COS-7 cells, primary cultures of rat anterior pituitary (AP) cells, the corticotroph-tumour cells AtT20 D16-16 and cortical neurons in primary culture. Our results indicate IR-CRH-R1 receptors have a punctate distribution on the plasma membrane of AP cells and AtT20 D16-16 cells. Whilst in AP cells their appearance is a fine punctate pattern, in AtT20 cells, they appear as large patches which could account for receptor clusters. Within primary cortical neurons, their distribution does not appear to be polarized. Our results suggest that distribution of CRH-R1 receptors within the different cell-types investigated depends not only on the amino acid sequence but also on cellular factors.

Prohormone and proneuropeptide synthesis and secretion.

Hormones and neuropeptides in eukaryotic cells, are synthesised as large precursor molecules in the rough endoplasmic reticulum (RER), from where they are translocated to the Golgi apparatus. The sorting of proteins destined for the regulated secretory pathway from those which will be released constitutively takes place in the trans-Golgi network (TGN). In both these pathways, vesicles need to be transported to the plasma membrane before their contents can be released by exocytosis. Hormones and neuropeptides need to be secreted from the cells in which are synthesised to exert their biological actions, although they can also play paracrine and autocrine actions. Prohormones and proneuropeptides must undergo post-translational modifications which occur in determined subcellular compartments within eukaryotic cells and are carried out in a strict succession of intracellular events, which give rise to biologically active products. The biosynthesis of prohormones/proneuropeptides is mediated by the action of endoproteolytic enzymes and other post-translational modifying enzymes within the secretory pathway. The major focus of this review will be the biosynthetic pathway, sorting and intracellular trafficking of prohormone and proneuropeptide precursors within the secretory pathway of eukaryotic cells.

Direct regulation of GnRH transcription by CRF-like peptides in an immortalized neuronal cell line.

The existence of a CRF-dependent inhibition of GnRH transcription was investigated using a neuronal GnRH-expressing cell line (Gn11) stably transfected with mouse (-611 bp) or chicken (-3000 bp) GnRH promoter/luciferase reporter constructs. The presence of the CRF-R1 receptor was established using a specific CRF-R1 antiserum. After 7 h of incubation, urotensin-I and sauvagine increased the mouse GnRH-reporter bioluminescence by 1.3- and 1.2-fold, respectively, compared with control cells. Subsequently, CRF, urotensin-I and sauvagine decreased luciferase reporter activity to about 60% of the control values after 14 h. Similar trends occurred with the chicken GnRH promoter with UI increasing reporter gene activity 2.4-fold over the controls after 14 h incubation. These data provide additional evidence for the direct regulation of GnRH transcription by CRF-like peptides.

Variable interval schedules of timeout from avoidance: effects of ethanol, naltrexone, and CGS 8216.

Four rats were trained on concurrent schedules of shock avoidance and timeout from avoidance, where responses on one lever postponed shock and responses on another lever occasionally (VI 45 sec schedule) produced a 2-min timeout during which the avoidance schedule was suspended. These procedures maintained stable rates of responding on both levers, providing a baseline for studying the effects of drugs on behavior under different types of aversive control (shock avoidance and timeout from avoidance). In the first experiment the effects of ethanol (0.5, 1.0, 1.5, and 2.0 g/kg) and an opiate antagonist, naltrexone (1 mg/kg) were assessed alone and in combination. Ethanol produced a dose-dependent decrease in avoidance characterized by increased shock rates and decreased response rates. At the same time, however, responding on the timeout lever generally increased relative to avoidance lever rates. All of these effects were largely confined to the early parts of the 2-hr session, when blood-ethanol levels were relatively high. Naltrexone had no effect on performances and did not interact with ethanol. In a second experiment, the effects of the benzodiazepine antagonist CGS 8216 were studied alone, and in combination with ethanol. CGS 8216 (5 mg/kg) generally disrupted both avoidance and timeout responding but did not reverse ethanol actions.

Variable-interval schedules of timeout from avoidance: effects of chlordiazepoxide, CGS 8216, morphine, and naltrexone.

Rats were trained on concurrent schedules in which pressing one lever postponed shock and pressing the other occasionally (variable-interval schedule) produced a 2-min timeout during which the shock-postponement schedule was suspended and its correlated stimuli were removed. These procedures provided a baseline for studying the effects of drugs on behavior maintained by different sources of negative reinforcement (shock avoidance and timeout from avoidance). Experiment 1 studied a benzodiazepine agonist, chlordiazepoxide, and antagonist, CGS 8216. Chlordiazepoxide (2.5-30 mg/kg) had little effect on avoidance responding except at higher doses, when it reduced responding. By comparison, responding on the timeout lever was increased in 5 of 6 rats. These effects were reversed by CGS 8216 (2.5-5 mg/kg) in the 2 rats tested, but CGS 8216 had no effect by itself. Experiment 2 studied an opiate agonist, morphine, and antagonist, naltrexone, with 3 rats. Morphine's (2.5-20 mg/kg) effects were opposite those of chlordiazepoxide: At doses that either increased or had no effect on avoidance responding, morphine depressed timeout responding. Naltrexone (5 mg/kg) reversed these actions but had no effect by itself.

Variable-interval schedules of timeout from avoidance.

Rats were trained on concurrent schedules in which pressing one lever postponed shock and pressing the other occasionally produced a 2-min timeout during which the shock-postponement schedule was suspended and its correlated stimuli were removed. Throughout, the shock-postponement schedule maintained proficient levels of avoidance. Nevertheless, in Experiment 1 responding on the timeout lever was established rapidly, was maintained at stable levels on variable-interval schedules, was extinguished by withholding timeout, was reestablished when timeout was reintroduced, and was brought under discriminative control with a multiple variable-interval extinction schedule of timeout. These results are in contrast with Verhave's (1962) conclusion that timeout is an ineffective reinforcer when presented to rats on intermittent schedules. In Experiment 2 the consequence of responding on the timeout lever was altered so that the shock-postponement schedule remained in effect even though the stimulus conditions associated with timeout were produced for 2 min. Responding extinguished, indicating that suspension of the shock-postponement schedule, not stimulus change, was the source of reinforcement. By establishing the reinforcing efficacy of timeout with standard variable-interval schedules, these experiments illustrate a procedure for studying negative reinforcement in the same way as positive reinforcement.

Fixed-ratio pausing: Joint effects of past reinforcer magnitude and stimuli correlated with upcoming magnitude.

Pigeons responded on fixed-ratio schedules ending in small or large reinforcers (grain presentations of different duration) interspersed within each session. In mixed-schedule conditions, the response key was lit with a single color throughout the session, and pausing was directly related to the past reinforcer (longer pauses after large reinforcers than after small ones). In multiple-schedule conditions, different colors accompanied the ratios ending in small and large reinforcers, and pausing was affected by the upcoming reinforcer as well as the past one. Pauses were shorter before large reinforcers than before small ones, but they continued to be longer after large reinforcers than after small ones. The influence of the past reinforcer was modulated by the magnitude of the upcoming reinforcer; in the presence of the stimulus before the small reinforcer, the effect of the past reinforcer was enhanced relative to its effect in the stimulus before the large reinforcer. These results show that pausing between ratios is jointly determined by two competing factors: past conditions of reinforcement and stimuli correlated with upcoming conditions.

Reductions in shock frequency and response effort as factors in reinforcement by timeout from avoidance.

Rats' presses on one lever canceled shocks programmed after variable cycles, while presses on a second lever occasionally produced a 2-min timout during which the shock-delection schedule was suspended and its correlated stimuli removed. These concurrent schedules of avoidance and timeout were embedded in a multiple schedule whose components differed, within and across conditions, in terms of the programmed shock rate associated with the shock-deletion schedule. Analyses based on the generalized matching law suggest that the reduction in the response requirement correlated with termination of the avoidance schedule was a more important factor in the reinforcing effectiveness of timeout than was shock-frequency reduction, at least in 2 of 3 rats. After training in each condition, responding on the timeout lever was extinguished by withholding timeouts in both components over seven sessions. Resistance to extinction varied directly with the rates of both shock-frequency reduction and avoidance-response reduction experienced during training. Although reduction in response effort appeared to dominate shock-frequency reduction in the maintenance of responding, neither factor had a clear advantage in predicting the course of extinction.