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Although hypercoagulability is commonly associated with malignancies, whether coagulation factors directly affect tumor cell proliferation remains unclear. Herein, by performing single-cell RNA sequencing (scRNA-seq) of the prostate tumor microenvironment (TME) of mouse models of castration-resistant prostate cancer (CRPC), we report that immunosuppressive neutrophils (PMN-MDSCs) are a key extra-hepatic source of coagulation factor X (FX). FX activation within the TME enhances androgen-independent tumor growth by activating the protease-activated receptor 2 (PAR2) and the phosphorylation of ERK1/2 in tumor cells. Genetic and pharmacological inhibition of factor Xa (FXa) antagonizes the oncogenic activity of PMN-MDSCs, reduces tumor progression, and synergizes with enzalutamide therapy. Intriguingly, F10 high PMN-MDSCs express the surface marker CD84 and CD84 ligation enhances F10 expression. Elevated levels of FX, CD84, and PAR2 in prostate tumors associate with worse survival in CRPC patients. This study provides evidence that FXa directly promotes cancer and highlights additional targets for PMN-MDSCs for cancer therapies., Competing Interests: Declaration of interests Authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cellular senescence can exert dual effects in tumors, either suppressing or promoting tumor progression. The senescence-associated secretory phenotype (SASP), released by senescent cells, plays a crucial role in this dichotomy. Consequently, the clinical challenge lies in developing therapies that safely enhance senescence in cancer, favoring tumor-suppressive SASP factors over tumor-promoting ones. Here, we identify the retinoic-acid-receptor (RAR) agonist adapalene as an effective pro-senescence compound in prostate cancer (PCa). Reactivation of RARs triggers a robust senescence response and a tumor-suppressive SASP. In preclinical mouse models of PCa, the combination of adapalene and docetaxel promotes a tumor-suppressive SASP that enhances natural killer (NK) cell-mediated tumor clearance more effectively than either agent alone. This approach increases the efficacy of the allogenic infusion of human NK cells in mice injected with human PCa cells, suggesting an alternative therapeutic strategy to stimulate the anti-tumor immune response in "immunologically cold" tumors., Competing Interests: Declaration of interests A.A. is a co-founder of and owns stock in OncoSense, and A.A., M.C., and A.R. are inventors of the patent WO2019142095A1 (Title: new alk inhibitor senolytic drugs)., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The human body hosts a complex and dynamic population of trillions of microorganisms - the microbiota - which influences the body in homeostasis and disease, including cancer. Several epidemiological studies have associated specific urinary and gut microbial species with increased risk of prostate cancer; however, causal mechanistic data remain elusive. Studies have associated bacterial generation of genotoxins with the occurrence of TMPRSS2-ERG gene fusions, a common, early oncogenic event during prostate carcinogenesis. A subsequent study demonstrated the role of the gut microbiota in prostate cancer endocrine resistance, which occurs, at least partially, through the generation of androgenic steroids fuelling oncogenic signalling via the androgen receptor. These studies present mechanistic evidence of how the host microbiota might be implicated in prostate carcinogenesis and tumour progression. Importantly, these findings also reveal potential avenues for the detection and treatment of prostate cancer through the profiling and modulation of the host microbiota. The latter could involve approaches such as the use of faecal microbiota transplantation, prebiotics, probiotics, postbiotics or antibiotics, which can be used independently or combined with existing treatments to reverse therapeutic resistance and improve clinical outcomes in patients with prostate cancer., (© 2023. Springer Nature Limited.)

Cancer is highly infiltrated by myeloid-derived suppressor cells (MDSCs). Currently available immunotherapies do not completely eradicate MDSCs. Through a genome-wide analysis of the translatome of prostate cancers driven by different genetic alterations, we demonstrate that prostate cancer rewires its secretome at the translational level to recruit MDSCs. Among different secreted proteins released by prostate tumor cells, we identified Hgf, Spp1 and Bgn as the key factors that regulate MDSC migration. Mechanistically, we found that the coordinated loss of Pdcd4 and activation of the MNK/eIF4E pathways regulate the mRNAs translation of Hgf, Spp1 and Bgn. MDSC infiltration and tumor growth were dampened in prostate cancer treated with the MNK1/2 inhibitor eFT508 and/or the AKT inhibitor ipatasertib, either alone or in combination with a clinically available MDSC-targeting immunotherapy. This work provides a therapeutic strategy that combines translation inhibition with available immunotherapies to restore immune surveillance in prostate cancer., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Androgen deprivation therapy (ADT) is a standard therapy for prostate cancer (PCa). Though disseminated disease is initially sensitive to ADT, an important fraction of the patients progresses to castration-resistant prostate cancer (CRPC). For this reason, the identification of novel effective therapies for treating CRPC is needed. Immunotherapeutic strategies focused on macrophages as antitumor effectors, directly enhancing their tumoricidal potential at the tumor microenvironment or their adoptive transfer after ex vivo activation, have arisen as promising therapies in several cancer types. Despite several approaches centered on the activation of tumor-associated macrophages (TAMs) in PCa are under investigation, to date there is no evidence of clinical benefit in patients. In addition, the evidence of the effectiveness of macrophage adoptive transfer on PCa is poor. Here we find that VSSP, an immunomodulator of the myeloid system, decreases TAMs and inhibits prostatic tumor growth when administered to castrated Pten-deficient prostate tumor-bearing mice. In mice bearing castration-resistant Pten pc-/- ; Trp53 pc-/- tumors, VSSP administration showed no effect. Nevertheless, adoptive transfer of macrophages activated ex vivo with VSSP inhibited Pten pc-/- ; Trp53 pc-/- tumor growth through reduction of angiogenesis and tumor cell proliferation and induction of senescence. Taken together, our results highlight the rationale of exploiting macrophage functional programming as a promising strategy for CRPC therapy, with particular emphasis on ex vivo-activated proinflammatory macrophage adoptive transfer. Video abstract., (© 2023. The Author(s).)

Tumor cells promote the recruitment of immunosuppressive neutrophils, a subset of myeloid cells driving immune suppression, tumor proliferation, and treatment resistance. Physiologically, neutrophils are known to have a short half-life. Here, we report the identification of a subset of neutrophils that have upregulated expression of cellular senescence markers and persist in the tumor microenvironment. Senescent-like neutrophils express the triggering receptor expressed on myeloid cells 2 (TREM2) and are more immunosuppressive and tumor-promoting than canonical immunosuppressive neutrophils. Genetic and pharmacological elimination of senescent-like neutrophils decreases tumor progression in different mouse models of prostate cancer. Mechanistically, we have found that apolipoprotein E (APOE) secreted by prostate tumor cells binds TREM2 on neutrophils, promoting their senescence. APOE and TREM2 expression increases in prostate cancers and correlates with poor prognosis. Collectively, these results reveal an alternative mechanism of tumor immune evasion and support the development of immune senolytics targeting senescent-like neutrophils for cancer therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cells subjected to treatment with anti-cancer therapies can evade apoptosis through cellular senescence. Persistent senescent tumor cells remain metabolically active, possess a secretory phenotype, and can promote tumor proliferation and metastatic dissemination. Removal of senescent tumor cells (senolytic therapy) has therefore emerged as a promising therapeutic strategy. Here, using single-cell RNA-sequencing, we find that senescent tumor cells rely on the anti-apoptotic gene Mcl-1 for their survival. Mcl-1 is upregulated in senescent tumor cells, including cells expressing low levels of Bcl-2, an established target for senolytic therapy. While treatment with the Bcl-2 inhibitor Navitoclax results in the reduction of metastases in tumor bearing mice, treatment with the Mcl-1 inhibitor S63845 leads to complete elimination of senescent tumor cells and metastases. These findings provide insights on the mechanism by which senescent tumor cells survive and reveal a vulnerability that can be exploited for cancer therapy., (© 2022. The Author(s).)

It has been recognized for decades that ERBB signaling is important in prostate cancer, but targeting ERBB receptors as a therapeutic strategy for prostate cancer has been ineffective clinically. However, we show here that membranous HER3 protein is commonly highly expressed in lethal prostate cancer, associating with reduced time to castration resistance (CR) and survival. Multiplex immunofluorescence indicated that the HER3 ligand NRG1 is detectable primarily in tumor-infiltrating myelomonocytic cells in human prostate cancer; this observation was confirmed using single-cell RNA sequencing of human prostate cancer biopsies and murine transgenic prostate cancer models. In castration-resistant prostate cancer (CRPC) patient-derived xenograft organoids with high HER3 expression as well as mouse prostate cancer organoids, recombinant NRG1 enhanced proliferation and survival. Supernatant from murine bone marrow-derived macrophages and myeloid-derived suppressor cells promoted murine prostate cancer organoid growth in vitro , which could be reversed by a neutralizing anti-NRG1 antibody and ERBB inhibition. Targeting HER3, especially with the HER3-directed antibody-drug conjugate U3-1402, exhibited antitumor activity against HER3-expressing prostate cancer. Overall, these data indicate that HER3 is commonly overexpressed in lethal prostate cancer and can be activated by NRG1 secreted by myelomonocytic cells in the tumor microenvironment, supporting HER3-targeted therapeutic strategies for treating HER3-expressing advanced CRPC. SIGNIFICANCE: HER3 is an actionable target in prostate cancer, especially with anti-HER3 immunoconjugates, and targeting HER3 warrants clinical evaluation in prospective trials., (©2021 The Authors; Published by the American Association for Cancer Research.)

The microbiota comprises the microorganisms that live in close contact with the host, with mutual benefit for both counterparts. The contribution of the gut microbiota to the emergence of castration-resistant prostate cancer (CRPC) has not yet been addressed. We found that androgen deprivation in mice and humans promotes the expansion of defined commensal microbiota that contributes to the onset of castration resistance in mice. Specifically, the intestinal microbial community in mice and patients with CRPC was enriched for species capable of converting androgen precursors into active androgens. Ablation of the gut microbiota by antibiotic therapy delayed the emergence of castration resistance even in immunodeficient mice. Fecal microbiota transplantation (FMT) from CRPC mice and patients rendered mice harboring prostate cancer resistant to castration. In contrast, tumor growth was controlled by FMT from hormone-sensitive prostate cancer patients and Prevotella stercorea administration. These results reveal that the commensal gut microbiota contributes to endocrine resistance in CRPC by providing an alternative source of androgens.

Metastases account for most cancer-related deaths, yet the mechanisms underlying metastatic spread remain poorly understood. Recent evidence demonstrates that senescent cells, while initially restricting tumorigenesis, can induce tumor progression. Here, we identify the metalloproteinase inhibitor TIMP1 as a molecular switch that determines the effects of senescence in prostate cancer. Senescence driven either by PTEN deficiency or chemotherapy limits the progression of prostate cancer in mice. TIMP1 deletion allows senescence to promote metastasis, and elimination of senescent cells with a senolytic BCL-2 inhibitor impairs metastasis. Mechanistically, TIMP1 loss reprograms the senescence-associated secretory phenotype (SASP) of senescent tumor cells through activation of matrix metalloproteinases (MMPs). Loss of PTEN and TIMP1 in prostate cancer is frequent and correlates with resistance to docetaxel and worst clinical outcomes in patients treated in an adjuvant setting. Altogether, these findings provide insights into the dual roles of tumor-associated senescence and can potentially impact the treatment of prostate cancer., Competing Interests: Declaration of Interests A.A. is a cofounder of and owns stock in OncoSense and is an inventor of the patent WO2019142095A1. E.S.A. is a paid consultant/advisor to Janssen, Astellas, Sanofi, Dendreon, Pfizer, Amgen, Eli-Lilly, Bayer, AstraZeneca, Bristol-Myers Squibb, Clovis, and Merck; he has received research funding to his institution from Janssen, Johnson & Johnson, Sanofi, Dendreon, Genentech, Novartis, Constellation, Bristol-Myers Squibb, AstraZeneca, Clovis, and Merck; and he is the co-inventor of a patented biomarker technology that has been licensed to QIAGEN., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Triple-negative breast cancer ( TNBC ) is a heterogeneous disease that lacks effective therapeutic options. In this study, we profile eighteen TNBC cell lines for their sensitivity to the anti-proliferative action of all-trans retinoic acid (ATRA). The only three cell lines ( HCC-1599 , MB-157 and MDA-MB-157 ) endowed with ATRA-sensitivity are characterized by genetic aberrations of the NOTCH1 -gene, causing constitutive activation of the NOTCH1 γ-secretase product, N1ICD. N1ICD renders HCC-1599 , MB-157 and MDA-MB-157 cells sensitive not only to ATRA, but also to γ-secretase inhibitors (DAPT; PF-03084014). Combinations of ATRA and γ-secretase inhibitors produce additive/synergistic effects in vitro and in vivo. RNA-sequencing studies of HCC-1599 and MB-157 cells exposed to ATRA and DAPT and ATRA+DAPT demonstrate that the two compounds act on common gene sets, some of which belong to the NOTCH1 pathway. ATRA inhibits the growth of HCC-1599 , MB-157 and MDA-MB-157 cells via RARα, which up-regulates several retinoid target-genes, including RARβ. RARβ is a key determinant of ATRA anti-proliferative activity, as its silencing suppresses the effects exerted by the retinoid. In conclusion, we demonstrate that ATRA exerts a significant anti-tumor action only in TNBC cells showing constitutive NOTCH1 activation. Our results support the design of clinical trials involving combinations between ATRA and γ-secretase inhibitors for the treatment of this TNBC subtype.

The mechanisms by which prostate cancer shifts from an indolent castration-sensitive phenotype to lethal castration-resistant prostate cancer (CRPC) are poorly understood. Identification of clinically relevant genetic alterations leading to CRPC may reveal potential vulnerabilities for cancer therapy. Here we find that CUB domain-containing protein 1 (CDCP1), a transmembrane protein that acts as a substrate for SRC family kinases (SFKs), is overexpressed in a subset of CRPC. Notably, CDCP1 cooperates with the loss of the tumor suppressor gene PTEN to promote the emergence of metastatic prostate cancer. Mechanistically, we find that androgens suppress CDCP1 expression and that androgen deprivation in combination with loss of PTEN promotes the upregulation of CDCP1 and the subsequent activation of the SRC/MAPK pathway. Moreover, we demonstrate that anti-CDCP1 immunoliposomes (anti-CDCP1 ILs) loaded with chemotherapy suppress prostate cancer growth when administered in combination with enzalutamide. Thus, our study identifies CDCP1 as a powerful driver of prostate cancer progression and uncovers different potential therapeutic strategies for the treatment of metastatic prostate tumors.

Tumors tissues house a diverse array of cell types, requiring powerful cell-based analysis methods to characterize cellular heterogeneity and identify rare cells. Tumor tissue is dissociated into single cells by treatment with proteolytic enzymes, followed by mechanical disruption using vortexing or pipetting. These procedures can be incomplete and require significant time, and the latter mechanical treatments are poorly defined and controlled. Here, we present a novel microfluidic device to improve mechanical dissociation of digested tissue and cell aggregates into single cells. The device design includes a network of branching channels that range in size from millimeters down to hundreds of microns. The channels also contain flow constrictions that generate well-defined regions of high shear force, which we refer to as "hydrodynamic micro-scalpels", to progressively disaggregate tissue fragments and clusters into single cells. We show using in vitro cancer cell models that the microfluidic device significantly enhances cell recovery in comparison to mechanical disruption by pipetting and vortexing after digestion with trypsin or incubation with EDTA. Notably, the device enabled superior results to be obtained after shorter proteolytic digestion times, resulting in fully viable cells in less than ten minutes. The device could also be operated under enzyme-free conditions that could better maintain expression of certain surface markers. The microfluidic format is advantageous because it enables application of well-defined mechanical forces and rapid processing times. Furthermore, it may be possible to directly integrate downstream processing and detection operations to create integrated cell-based analysis platforms. The enhanced capabilities enabled by our novel device may help promote applications of single cell detection and purification techniques to tumor tissue specimens, advancing the current understanding of cancer biology and enabling molecular diagnostics in clinical settings.

Objective: This study investigated family functioning in adolescents with the restricting and bulimic type of anorexia nervosa (AN) and in healthy controls., Method: Fifty-one parents and their children (17 with AN and 34 healthy adolescents) completed the Family Assessment Measure, a self-report instrument that provides information about the functional strengths and weaknesses of the family and each family member., Results: AN patients with the bulimic subtype and their mothers were significantly more likely to perceive family functioning as impaired than were healthy adolescents or restricting AN patients. Restricting AN patients and their families did not differ from healthy control families., Conclusion: The results suggest that the problems faced by bulimic patients color their and their parents' view of each other and the family. Symptomatic and personality differences between the subtypes of AN with better control, fewer symptoms, and denial of conflict characterizing restricting AN families may be reflected in family interaction styles.

Objective: To actively detect leishmaniasis in HIV-1-infected individuals in Italy, to describe the epidemiological features of the disease in these patients, and to compare them with epidemiological features of leishmaniasis in HIV-negative patients., Design: Retrospective and prospective surveillance study., Patients: Italian patients with HIV-1 infection and leishmaniasis diagnosed between 1985 and 1994., Results: We recorded 116 leishmaniasis cases (115 visceral leishmaniasis), of which 94 (81%) were diagnosed over the last 4 years. Seventy-eight patients (67%) fulfilled the 1993 Centers for Disease Control and Prevention AIDS criteria. Leishmaniasis was passively reported in only 18% of cases. Leishmania incidence estimated among approximately 2700 AIDS patients living in leishmaniasis endemic areas averaged 1.6%, with a maximum of 4.9% in Sicily. These rates were up to 500-fold higher than among HIV-negative individuals living in the same areas, and were similar to those of ubiquitous opportunistic agents indicative of AIDS condition. Data from two major endemic regions indicated that overlap of HIV-1 and Leishmania infections has focal characteristics. The occurrence of small case clusters would suggest occasional modes of Leishmania transmission different from the insect vector. The isoenzyme characterization of 38 Leishmania stocks showed a zymodeme spectrum qualitatively and quantitatively different from that of the parasitic agent of visceral leishmaniasis in HIV-negative adults., Conclusions: Active surveillance provided reliable evaluation on the occurrence of HIV-Leishmania coinfections in Italy, although it was limited to hospital-based cases in this study due to general under-reporting of cases. Biological and epidemiological spectrum of the disease suggests that visceral leishmaniasis should be included among AIDS-defining pathologies.

Eleven zymodemes of Leishmania infantum were identified among 38 parasite stocks isolated from Italian HIV-positive patients with visceral leishmaniasis (VL). Only one zymodeme is a common agent of Mediterranean VL in HIV-negative individuals, five zymodemes usually cause simple, self-resolving cutaneous leishmaniasis (CL), and five belong to unique genotypes which have not been previously reported from either VL or CL cases in immunocompetent individuals. This last group of parasites showed reassortment patterns within electromorphs frequently observed in dermotropic L. infantum zymodemes. The highest zymodeme heterogeneity was found in south Italy (Sicily), with six zymodemes identified among 12 HIV-positive patients surveyed.

The antidepressant trazodone and its main metabolite, m-CPP, having an antiserotoninergic and serotoninergic activity respectively, were studied in an acute dependence model in mice, to establish whether 5-hydroxytryptaminergic systems are involved in the manifestations of acute opiate dependence and in its development. When drugs were administered 15 min before naloxone, all signs of abstinence decreased, with the exception of teeth chattering that was increased by m-CPP and unaffected by trazodone. When injected 15 min before morphine, jump episodes were decreased by the highest doses of both drugs, while teeth chattering was decreased by m-CPP only. When administered 1 h before morphine, trazodone increased paw and head shakes and mCPP decreased teeth chattering and both left the other signs unaffected. Serotoninergic systems seem to have a significant role in events involved in the withdrawal syndrome and a minor one in those leading to the development of dependence.

The authors examine 152 cases of femoral neck fractures treated with Knowles pins in order to analyze the incidence and causes of nonunion and avascular necrosis. The displaced fractures were all treated with closed reduction, and weight-bearing was permitted after 30-45 days. Seventy fractures were followed up after an average of four years. Criteria are proposed for evaluating the quality of both the reduction and the internal fixation. Healing was achieved in all cases of Garden grade I and II fractures; the incidence of avascular necrosis was 4.1%. In the displaced fractures the incidence of nonunion was 15.2% and avascular necrosis 13%. Technical errors in either reduction or internal fixation were present in 50% of the necroses and in 100% of the nonunions. The quality of the reduction was the most important factor in fracture healing, yet an accurate reduction and a valid fixation could not completely overcome the risk of avascular necrosis in the femoral head.

The nocturnal pattern of Syrian hamster pineal melatonin synthesis is characterized by a 6-8 h lag period, followed by a late-night, short-duration peak in both N-acetyltransferase (NAT) activity and melatonin content. Administration of cycloheximide (20 mg/kg body weight) given either at the time of lights out or 4 h into the dark phase to Syrian hamsters blocked the nocturnal increase in both pineal NAT activity and melatonin content. Actinomycin D (5 mg/kg body weight) prevented the nocturnal increase in both constituents only when it was administered at darkness onset, being significantly less effective when injected after 4 h of dark exposure. Reinduction of melatonin production by isoproterenol (2 mg/kg body weight) administration to acutely light-exposed animals during late darkness was prevented by cycloheximide, but not by actinomycin D administration. The results suggest that whereas Syrian hamster pineal melatonin production requires protein synthesis both early and late in the dark phase, the transcription of a putative NAT-related mRNA, which occurs only during the early night, seems to determine the lag period in melatonin synthesis and pineal responsiveness to beta-adrenergic receptor agonist stimulation.

The Harderian glands of Syrian hamsters contain melatonin and the enzymes N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) which synthesize melatonin from serotonin. Because the Harderian glands share this metabolic pathway with the pineal gland, we examined the effects of short-day photoperiods, which stimulate pineal-mediated gonadal regression, and N-(2,4-dinitrophenyl)-5-methoxytryptamine (ML-23), which has been described as a melatonin antagonist, on melatonin synthesis in the Harderian glands of the hamster. Harderian glands of male hamsters kept in short days had reduced NAT activity and melatonin concentration, but HIOMT activity was unchanged from that of long-day controls. In males kept in short days, ML-23 restored melatonin concentrations to levels seen in long days but did not affect the short-day induced reduction in NAT activity. ML-23 had no effect upon NAT or HIOMT activity or melatonin concentration in male hamsters kept on long days. Harderian glands of female hamsters kept on short days had reduced melatonin concentrations, but NAT and HIOMT activities similar to those of long-day controls. ML-23 had no effect on Harderian NAT or HIOMT activities or melatonin concentration in females kept in short days. However, in females kept in long days, ML-23 treatment led to increased NAT activity and decreased melatonin concentrations. We conclude from these results that short-day photoperiods alter some aspects of melatonin synthesis in hamster Harderian glands and that these effects differ in males and females. ML-23 does not usually prevent the effects of short days on Harderian melatonin synthesis, suggesting that it is not a melatonin antagonist in the Syrian hamster.

A naturally occurring compound, 6-methoxybenzoxazolinone (6-MBOA), present in grasses, has been shown to induce sexual maturation in a number of rodent species. The structural similarity of 6-MBOA and melatonin has led researchers to suspect that 6-MBOA might induce its progonadal effects by directly altering pineal function. Previous studies have shown that 6-MBOA has the properties of a weak beta-adrenergic agonist capable of stimulating rat pineal N-acetyltransferase (NAT) activity at pharmacological concentrations of 10(-3) M. In the present study we have examined the effect of 6-MBOA on both the pineal melatonin synthesizing enzymes, namely, NAT and hydroxyindole-O-methyltransferase (HIOMT) as well as on melatonin production, in organ cultured rat pineal glands. In addition, we have also examined the ability of 6-MBOA to displace a ligand from rat brain alpha- and beta-adrenoceptors. Our results confirm that 6-MBOA stimulates NAT activity and melatonin production at the high concentration of 10(-3) M. It appears to have no effect on HIOMT activity. A competition study shows that 6-MBOA is able of displacing ligands at the alpha- and beta-adrenoceptors but only at concentrations greater than 10(-4) M. Whether such high concentrations of 6-MBOA reach the pineal of rodent in their natural habitat is unknown. However, if 6-MBOA does mediate progonadal effects by altering pineal function it would be expected that 6-MBOA would ultimately inhibit the effects of melatonin. The possibilities are that the high melatonin levels induced by 6-MBOA cause desensitization of melatonin receptors or that 6-MBOA is an antagonist at the level of the melatonin receptor, thus restricting the inhibitory effects of melatonin on the reproductive system.

Melatonin synthesis in the pineal gland, which is primarily regulated by the environmental lighting regime, can also be influenced by other factors that elicit modifications in sympathetic tone. The objectives of this study were to determine if forced swimming alters the normal pattern of melatonin production in the pineal gland of the Richardson's ground squirrel (Spermophilus richardsonii). In early June, the squirrels were forced to swim for 10 min during the photophase or during the scotophase. In mid-July squirrels swam only during the scotophase. Animals were sacrificed 15, 30, or 60 min after the onset of swimming. Activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were assessed by radioenzyme assay, and pineal melatonin content was measured by radioimmunoassay. Daytime swimming elicited no major changes in enzyme activity or pineal melatonin. In June, swimming at night prevented the normal rises in NAT activity and pineal melatonin seen in nonswimming controls. In contrast, the pineals of squirrels that were tested 6 weeks later in mid-July did not appear to be as sensitive to nighttime swimming, as there were only minor differences in both NAT activity and melatonin content compared to controls. These results demonstrate that forced nighttime swimming, unlike several other aversive stimuli, can evoke changes in the normal pattern of pineal melatonin production in this species. Furthermore, the pineal's response to such stimuli may not be stable over the course of the active season.

In light of recent studies demonstrating stress-induced changes in pineal indoleamine metabolism, we tested the effect of acute cold stress on pineal biosynthetic function. Adult male rats were subjected to 30, 60, or 120 min of cold exposure (Ta = 2 degrees C) during either the light or dark phase of the daily photoperiodic cycle. Controls were kept at room temperature (22 +/- 2 degrees C). Animals were killed by decapitation and pineals were analyzed by radioimmunoassay for melatonin content and by radioenzymeassay for the activity of N-acetyltransferase (NAT). Cold exposure during the day elicited no significant changes in pineal indoleamine metabolism. Exposure to cold for 1 hr during the second hour after lights off slightly increased pineal melatonin content, without a concomitant change in NAT activity. Rats exposed to 2 hr of cold beginning 2 hr after lights off, however, displayed a 50% reduction in NAT activity, whereas pineal melatonin content remained unchanged. The paradoxical response of pineal NAT activity and melatonin content are not uncommon when rats are exposed to adverse stimuli.

Since the pineal gland is an end organ of the sympathetic nervous system, stress might increase the synthesis of its hormone, melatonin. The stress of a 10 min swim, which elicits a marked rise in circulating catecholamines, causes a dramatic depression of high pineal melatonin levels at night within 15 min after swimming onset. N-acetyltransferase (NAT) activity is unaffected by the treatment at 15 or 30 min after swimming onset. Within 90 min after initiation of a 15 min swim, high nighttime pineal melatonin levels are restored while NAT values remain elevated. The swimming-induced reduction in high pineal melatonin levels is not influenced by either hypophysectomy, superior cervical ganglionectomy, prazosin (alpha 1-adrenergic receptor blocker) pretreatment, yohimbine (alpha 2-adrenergic receptor blocker) pretreatment, or reserpine (amine depletor) pretreatment. These results indicate that neither hormones secreted from the pituitary gland nor catecholamines secreted from the sympathetic nerves are involved in eliciting the dramatic reduction in elevated pineal melatonin levels in the rat.

A hind-leg subcutaneous saline injection into rats at night elicits a decrease in N-acetyltransferase (NAT) activity and melatonin content of the pineal gland. The decrement in pineal melatonin production after saline injection is prevented by adrenalectomy. The present studies were undertaken to determine what factor(s) from the adrenal gland cause(s) the drop in pineal melatonin production after saline injection at night. In the first study, groups of intact and adrenal-demedullated male rats were given a saline injection at 23.10 h (3 h, 10 min after lights off) and their pineals were collected 15 or 30 min later. Pineal NAT activity was depressed in both intact and adrenal-demedullated rats at 15 min postinjection as compared to their respective control animals. Pineal melatonin levels exhibited a drop in intact animals at 15 min and in adrenal-demedullated rats at 30 min. In a second study, hypophysectomy was found to prevent the drop in nocturnal pineal NAT activity and melatonin levels normally associated with a hind leg injection of saline. Finally, in a third experiment, groups of hypophysectomized rats were injected i.p. with corticosterone at 23.10 h and killed 10, 25 or 40 min postinjection. Corticosterone injection in hypophysectomized rats produced a response similar to that caused by saline injection in intact animals: NAT activity was depressed at 10 min and melatonin content was lowered at 25 min. These results suggest that the adrenal-mediated depression in melatonin synthesis after saline injection at night in rats may be elicited by an adrenal cortical hormone (corticosterone) and apparently does not involve the release of factors from the adrenal medulla.

To determine if exogenously administered alpha-melanocyte stimulating hormone (alpha-MSH) affects nighttime pineal N-acetyltransferase activity, pineal levels of 5-hydroxytryptophan, serotonin and melatonin, and plasma prolactin levels, adult male hamsters were injected at 1900 hr (lights out 2000-0600 hr) with two doses of the peptide and killed at 0300 hr. The low dose of alpha-MSH (200 ng) produced a significant fall in pineal serotonin, pineal NAT activity and plasma prolactin values. The high dose of the peptide (20 micrograms) increased circulating prolactin titers and pineal serotonin levels and caused a concomitant decrease in pineal melatonin levels.

Isoproterenol (1 mg/kg) was subcutaneously injected into adult male rats during the day to stimulate pineal N-acetyltransferase (NAT) activity and pineal and serum melatonin levels. Two hours after isoproterenol administration when levels of each of these variables had increased significantly, the experimental animals swam for 10 min in 22 degrees C water. At 15 min after swimming onset, pineal and serum melatonin levels were highly significantly depressed compared to those in control animals that did not swim. The high NAT level was not influenced by swimming. In a second study, isoproterenol injected rats swam for either 1, 3, 6 or 10 min and were sampled 15 min after the onset of swimming. The reduction in the elevated pineal melatonin in these animals was correlated with the length of the swim, i.e., as the duration of swim increased the percent reduction in pineal melatonin also increased. Neither pineal NAT nor hydroxyindole-O-methyltransferase (HIOMT) activities were influenced by swimming. The results suggest that elevated pineal and serum melatonin induced by isoproterenol can be depressed with no effect on the activity of the enzymes which convert serotonin to melatonin.

N-acetyltransferase (NAT) is believed to be the rate-limiting enzyme in the synthesis of melatonin from serotonin in the pineal gland. Norepinephrine released from sympathetic nerve endings within the pineal gland stimulates NAT activity and, therefore, melatonin synthesis. When an animal is subjected to a stressful stimulus, it would be expected that the increase in plasma stimulus, it would be expected that the increase in plasma catecholamines originating from the adrenal medulla and/or the sympathetic nervous system would result in a stimulation of pineal NAT activity. Adult male rats were given a 1.5cc injection of physiological saline subcutaneously into the back leg. Compared to non-injected controls, animals stressed in this manner were shown to have significantly lower pineal melatonin content 10 min after the saline injection late in the light phase of the light/dark cycle (at 18.30 h-lights on at 07.00 h). To test this more thoroughly, a time course study was conducted during the dark phase (at 02.00 h-5 hours after lights out) when pineal NAT activity and melatonin levels are either increasing or elevated. NAT activity and melatonin levels in the pineal were significantly depressed in stressed animals as compared to controls by 10 min after the saline injection, and remained so until 60 min after injection. By 90 min they had returned to control values. In the next study the nighttime response of the pineal to stress was compared in intact and adrenalectomized rats. Adrenalectomy prevented the changes in NAT activity and melatonin content associated with the saline injection. Some factor, such as a catecholamine or corticosterone from the adrenal, seems to be eliciting the response in the pineal to the saline injection. It is not known if the factor is acting centrally or directly on the pineal gland.

Seven experiments were performed to investigate the sensitivity of the hamster pineal gland to exogenously administered norepinephrine (NE). In these studies NE (1 mg/kg) administration was preceded (10 min earlier) by the injection of the catecholamine uptake inhibitor desmethylimipramine (DMI; 5 mg/kg). When DMI and NE were given at night, the hamsters were exposed to light to depress pineal N-acetyltransferase activity and melatonin values to low levels; the drugs were then given 20 (DMI) and 30 (NE) min later, and the subsequent changes in pineal N-acetyltransferase and melatonin were monitored. The combination of DMI and NE administration anytime during the normal light period or during the first 4 h of the normal dark period failed to stimulate either pineal N-acetyltransferase activity or melatonin levels. Conversely, DMI followed by NE (injected either intraperitoneally or subcutaneously) in the second half of the dark phase typically stimulated pineal melatonin production. Likewise, the NE agonist isoproterenol promoted pineal melatonin production only in the latter half of the dark phase. If hamsters were exposed to continual light at night or if they were superior cervical ganglionectomized, a procedure which sympathetically denervates the pineal gland, the stimulatory effect of NE on melatonin production was significantly suppressed. Thus, the hamster pineal gland is sensitive to NE only during the latter half of the normal dark period and both darkness and an intact sympathetic innervation to the pineal gland are required for the gland to develop maximal sensitivity to the catecholamine. Also, the hamster pineal seems not to exhibit a supersensitivity response to NE following a period of reduced exposure to the catecholamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult male Syrian (golden) hamsters, maintained under either 22 +/- 2 or 32 +/- 2 degrees C, were treated with 8 or 11 weeks of exposure to either long photoperiod (14:10), short photoperiod (8:16), or to long photoperiod with a daily afternoon melatonin injection. By 8 weeks, the animals kept at 22 degrees C and treated with daily afternoon melatonin injection exhibited a dramatic reduction in testicular and accessory sex organ weight, but the animals kept at 32 degrees C and treated in the same way exhibited only slight decreases in testicular and accessory organ weights. Short photoperiod caused a slight decrease in testicular and accessory organ weights of hamster kept at 22 degrees C, while it had no significant effects on reproductive organ weights of the animals maintained under 32 degrees C. By 11 weeks, the daily afternoon melatonin injection elicited further reduction in testicular and accessory organ weights of the animals maintained under both 22 and 32 degrees C. However, the reduction in animals kept at 32 degrees C was not as great as that in animals kept at 22 degrees C. Although short photoperiod caused an obvious decline in reproductive organ weights of the animals at 22 degrees C, only a slight decrease was seen in hamsters at 32 degrees C. As with reproductive organ weights, testosterone levels were depressed more rapidly and completely in animals maintained at 22 degrees C. These results indicate that elevated ambient temperature changes the rate at which the gonads of hamsters regress in response to daily afternoon melatonin injections or short photoperiod.(ABSTRACT TRUNCATED AT 250 WORDS)

The activities of NAT and HIOMT and the melatonin concentration in the Harderian glands of intact, gonadectomized, and gonadally-regressed male Syrian hamsters were studied. To produce gonadal regression, hamsters were exposed to either artificial or naturally short photoperiods. NAT activity of castrated and gonadally-regressed hamsters was always less in comparison to that of animals with intact gonads. Castrated hamsters exposed to long days showed higher NAT activity than that of castrated animals exposed to short photoperiods indicating that light may have some influence on Harderian NAT independent of the gonadal status. Also, gonadally-regressed hamsters exposed to long photoperiods exhibited higher NAT activity in comparison to gonadally-regressed animals exposed to short days. The HIOMT activity and melatonin content of Harderian glands in all these groups of male Syrian hamster were very low.

The role of cyclic AMP in the regulation of melatonin production was investigated in cultured Syrian hamster pineal glands. Forskolin markedly increased cyclic AMP production in pineal glands collected either late in the light period or in the dark period. The effect of forskolin was synergistically enhanced by 3-isobutylmethylxanthine, a phosphodiesterase inhibitor; however, increase in cyclic AMP after isoproterenol was only apparent in the presence of 3-isobutylmethylxanthine. Since beta-adrenergic agonists are able to stimulate melatonin production late in the dark period only, these data suggest that, in the hamster pineal gland, there may be intracellular mechanisms in addition to a cyclic AMP increase required for induction of melatonin production by beta-adrenergic agonists.

The nocturnal rises in pineal N-acetyltransferase (NAT) activity and melatonin levels were compared in young (25-35 days old) and adult (at least 1 year old) Richardson's ground squirrels. When expressed as NAT activity per pineal gland, the nighttime rise in the activity of this enzyme was less in young than in the adult animals; conversely, the melatonin content of the pineal glands of young animals was higher at one point (4 a.m., 8 hours after darkness onset) when compared to that in adult squirrels. When data were expressed relative to total protein, the NAT and melatonin rhythms in the pineals of young and adult animals were very similar. The effect of insulin-induced hypoglycemia on both daytime and nighttime NAT and melatonin levels in the pineal gland of the Richardson's ground squirrel was also assessed. Low daytime levels of these constituents were not influenced by the administration of 10 units insulin, a treatment which caused a marked drop in circulating glucose levels. At night, when pineal NAT and melatonin levels were high insulin injection had a very modest stimulatory effect on NAT activity (one point was elevated above saline injected controls) while melatonin levels remained unchanged by the treatment. These findings in the ground squirrel in reference to insulin-induced hypoglycemia, and stressors in general, appear to differ from those in the rat where stress can have a substantial influence or both low daytime and high nighttime levels of pineal NAT and melatonin.

Recently, it was shown that a 1.5-ml subcutaneous saline injection depressed N-acetyltransferase (NAT) activity and melatonin content in the rat pineal gland at night. The present studies were undertaken to determine if another perturbation, swimming, could duplicate this response. Rats swam at 23.10 h (lights out at 20.00 h) for 10 min and were killed 15 and 30 min after the unset of swimming. Pineal NAT activity was found to be unaffected while melatonin content was depressed dramatically. Hydroxyindole-O-methyltransferase (HIOMT) activity as well as the content of serotonin (5HT), 5-hydroxytryptophan (5HTP) and 5-hydroxyindoleacetic acid (5HIAA) were not changed by this treatment. In a second study, pineal melatonin again was depressed without a concomitant drop in NAT activity. Mean serum melatonin at 15 min after onset of swimming was increased although the rise was not statistically significant. In the final study, it was found that NAT activity was slightly increased in intact rats and unchanged in adrenalectomized rats at 7 min after swimming onset. At 15 min both intact and adrenalectomized animals had NAT activity values similar to those of controls. Pineal melatonin content in intact and adrenalectomized rats plummeted to 50% of control values at 7 min and fell further to 25% at 15 min. While the rate of melatonin synthesis was not directly measured, lack of change in the activities of the enzymes involved in melatonin synthesis and the contents of two melatonin precursors suggests that swimming depresses pineal melatonin content by enhancing melatonin efflux from the gland.

The activity of N-acetyltransferase (NAT) and the content of melatonin (MEL) in the rat pineal have been shown to be sensitive to several types of stressors. This study was designed to assess the role of the adrenals in mediating the effect of one such stressor, insulin-induced hypoglycemia, on pineal synthetic activity. Intact and bilaterally adrenalectomized (ADX) adult male rats were kept under light:dark cycles of 14:10 (lights on 0600 h) and injected intraperitoneally with 10 IU insulin at 1300 h, and groups (n = 8) were killed 2, 3, or 4 h postinjection. Plasma catecholamines were assayed by means of high performance liquid chromatography and radioimmunoassay was used to assess pineal NAT activity and MEL content. All injected groups were rendered hypoglycemic by insulin administration. Compared to uninjected controls, plasma epinephrine in hypoglycemic intact rats rose after 2 h, whereas epinephrine did not change in hypoglycemic ADX animals. The increase in epinephrine in intact animals was correlated with a rise in NAT activity at 2 h. Moreover, pineal MEL content at 2, 3, and 4 h was significantly greater than control values. In contrast, no changes in pineal biosynthetic function were found in ADX rats. This differential response by intact and ADX rats suggests that an adrenal product (possibly epinephrine) is responsible for mediating the stimulatory effects of acute insulin-induced hypoglycemic stress on the rat pineal.

The influence of delta-sleep inducing peptide (DSIP) on rat pineal N-acetyltransferase (NAT) activity and melatonin levels was examined. Young adult rats received an injection of either saline or DSIP (either 15, 30 or 60 nmol/kg) at 20:00 h, immediately before lights out. DSIP at a dose of 15 nmol/kg significantly retarded the nighttime rise of pineal NAT activity and melatonin levels at 3 h into the dark phase. At the other two nocturnal time points (01:00 and 03:00 h) and the following morning (08:00 h) pineal NAT activity and melatonin levels were similar in all groups. The results confirmed a short-lived, dose-specific effect of DSIP on serotonin metabolism in the rat pineal gland.