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TRH is synthesized from a larger 26-kilodalton (kDa) prohormone (pro-TRH). Rat pro-TRH contains five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven other cryptic peptides. Each of the five TRH progenitor sequences is flanked by pairs of basic amino acids. We used a bovine intermediate lobe secretory vesicle membrane preparation, which contains the prohormone convertases (PCs) PC1 and PC2, to study the in vitro processing of pro-TRH. Pro-TRH was radiolabeled using [3H]Leu in AtT20 cells transfected with prepro-TRH complementary DNA, and the labeled 26-kDa pro-TRH was isolated from the cell extract by preparative sodium dodecyl sulfate-gel electrophoresis. Incubation of [3H]pro-TRH with the intermediate lobe secretory vesicle membrane preparation was followed by immunoprecipitation with antibodies specific for various regions of the pro-TRH sequence, and the immunoprecipitates were analyzed by sodium dodecyl sulfate-gel electrophoresis. Immunoprecipitation of the reaction mixture with anti-pCC10 antibody (an antibody that recognizes the intact precursor and amino-terminal intermediate products of processing) showed a time-dependent appearance of a 15-kDa and a 6-kDa peptide and, at times, a 3.8-kDa peptide with diminution of the 26-kDa substrate. Immunoprecipitation of the incubate with the C-terminal-directed antibody, pYE17 (an antibody that recognizes the intact precursor and C-terminal intermediate products of processing), showed the generation of 16.5-, 10-, and 5.4-kDa products in a time-dependent manner, with disappearance of the substrate. Western blot analysis demonstrated that the secretory vesicle membrane preparation contains PC1 and PC2. Immunodepletion studies with antiserum specific for PC1 or PC2 demonstrated that PC1 and PC2 can process pro-TRH to these intermediate products. An initial site of cleavage appeared to be either at the 152-153 or the 158-159 pair of basic residues to yield a 15-kDa N-terminal fragment that was then processed to the 6-kDa [TRH-(25-74)] and 3.8-kDa [TRH-(83-112)] forms. The 10-kDa C-terminal peptide generated by this cleavage was then processed to a 5.4-kDa peptide [TRH-(208-255)]. Alternatively, an initial cleavage at the 107-108 or the 112-113 bonds was also observed, yielding a 16.5-kDa C-terminal product that was further processed to the 5.4-kDa peptide. The pH profile for the appearance of both C- and N-terminal products showed a bimodal distribution, with optima at both 5.5 and 7.5. The cleavage of pro-TRH was enhanced by Ca2+ and partially inhibited by Zn2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Purpose: Innate immune activation plays a critical role in the onset and progression of many diseases. While positron emission tomography (PET) imaging provides a non-invasive means to visualize and quantify such immune responses, most available tracers are not specific for innate immune cells. To address this need, we developed [ 18 F]OP-801 by radiolabeling a novel hydroxyl dendrimer that is selectively taken up by reactive macrophages/microglia and evaluated its ability to detect innate immune activation in mice following lipopolysaccharide (LPS) challenge., Procedures: OP-801 was radiolabeled in two steps: [ 18 F]fluorination of a tosyl precursor to yield [ 18 F]3-fluoropropylazide, followed by a copper-catalyzed click reaction. After purification and stability testing, [ 18 F]OP-801 (150-250 μCi) was intravenously injected into female C57BL/6 mice 24 h after intraperitoneal administration of LPS (10 mg/kg, n=14) or saline (n=6). Upon completing dynamic PET/CT imaging, mice were perfused, and radioactivity was measured in tissues of interest via gamma counting or autoradiography., Results: [ 18 F]OP-801 was produced with >95% radiochemical purity, 12-52 μCi/μg specific activity, and 4.3±1.5% decay-corrected yield. Ex vivo metabolite analysis of plasma samples (n=4) demonstrated high stability in mice (97±3% intact tracer >120 min post-injection). PET/CT images of mice following LPS challenge revealed higher signal in organs known to be inflamed in this context, including the liver, lung, and spleen. Gamma counting confirmed PET findings, showing significantly elevated signal in the same tissues compared to saline-injected mice: the liver (p=0.009), lung (p=0.030), and spleen (p=0.004). Brain PET/CT images (summed 50-60 min) revealed linearly increasing [ 18 F]OP-801 uptake in the whole brain that significantly correlated with murine sepsis score (r=0.85, p<0.0001). Specifically, tracer uptake was significantly higher in the brain stem, cortex, olfactory bulb, white matter, and ventricles of LPS-treated mice compared to saline-treated mice (p<0.05)., Conclusion: [ 18 F]OP-801 is a promising new PET tracer for sensitive and specific detection of activated macrophages and microglia that warrants further investigation., (© 2023. The Author(s), under exclusive licence to World Molecular Imaging Society.)

Chronic innate immune activation is a key hallmark of many neurological diseases and is known to result in the upregulation of GPR84 in myeloid cells (macrophages, microglia, and monocytes). As such, GPR84 can potentially serve as a sensor of proinflammatory innate immune responses. To assess the utility of GPR84 as an imaging biomarker, we synthesized 11 C-MGX-10S and 11 C-MGX-11S via carbon-11 alkylation for use as positron emission tomography (PET) tracers targeting this receptor. In vitro experiments demonstrated significantly higher binding of both radiotracers to hGPR84-HEK293 cells than that of parental control HEK293 cells. Co-incubation with the GPR84 antagonist GLPG1205 reduced the binding of both radiotracers by >90%, demonstrating their high specificity for GPR84 in vitro . In vivo assessment of each radiotracer via PET imaging of healthy mice illustrated the superior brain uptake and pharmacokinetics of 11 C-MGX-10S compared to 11 C-MGX-11S . Subsequent use of 11 C-MGX-10S to image a well-established mouse model of systemic and neuro-inflammation revealed a high PET signal in affected tissues, including the brain, liver, lung, and spleen. In vivo specificity of 11 C-MGX-10S for GPR84 was confirmed by the administration of GLPG1205 followed by radiotracer injection. When compared with 11 C-DPA-713-an existing radiotracer used to image innate immune activation in clinical research studies- 11 C-MGX-10S has multiple advantages, including its higher binding signal in inflamed tissues in the CNS and periphery and low background signal in healthy saline-treated subjects. The pronounced uptake of 11 C-MGX-10S during inflammation, its high specificity for GPR84, and suitable pharmacokinetics strongly support further investigation of 11 C-MGX-10S for imaging GPR84-positive myeloid cells associated with innate immune activation in animal models of inflammatory diseases and human neuropathology., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Positron emission tomography (PET) is a powerful tool for studying neuroinflammatory diseases; however, current PET biomarkers of neuroinflammation possess significant limitations. We recently reported a promising dendrimer PET tracer ([ 18 F]OP-801), which is selectively taken up by reactive microglia and macrophages. Here, we describe further important characterization of [ 18 F]OP-801 in addition to optimization and validation of a two-step clinical radiosynthesis. [ 18 F]OP-801 was found to be stable in human plasma for 90 min post incubation, and human dose estimates were calculated for 24 organs of interest; kidneys and urinary bladder wall without bladder voiding were identified as receiving the highest absorbed dose. Following optimization detailed herein, automated radiosynthesis and quality control (QC) analyses of [ 18 F]OP-801 were performed in triplicate in suitable radiochemical yield (6.89 ± 2.23% decay corrected), specific activity (37.49 ± 15.49 GBq/mg), and radiochemical purity for clinical imaging. Importantly, imaging mice with tracer (prepared using optimized methods) 24 h following the intraperitoneal injection of liposaccharide resulted in the robust brain PET signal. Cumulatively, these data enable clinical translation of [ 18 F]OP-801 for imaging reactive microglia and macrophages in humans. Data from three validation runs of the clinical manufacturing and QC were submitted to the Food and Drug Administration (FDA) as part of a Drug Master File (DMF). Subsequent FDA approval to proceed was obtained, and a phase 1/2 clinical trial (NCT05395624) for first-in-human imaging in healthy controls and patients with amyotrophic lateral sclerosis is underway.

Intro: Chronic neuroinflammation and microglial dysfunction are key features of many neurological diseases, including Alzheimer's Disease and multiple sclerosis. While there is unfortunately a dearth of highly selective molecular imaging biomarkers/probes for studying microglia in vivo, P2Y12R has emerged as an attractive candidate PET biomarker being explored for this purpose. Importantly, P2Y12R is selectively expressed on microglia in the CNS and undergoes dynamic changes in expression according to inflammatory context (e.g., toxic versus beneficial/healing states), thus having the potential to reveal functional information about microglia in living subjects. Herein, we identified a high affinity, small molecule P2Y12R antagonist (AZD1283) to radiolabel and assess as a candidate radiotracer through in vitro assays and in vivo positron emission tomography (PET) imaging of both wild-type and total knockout mice and a non-human primate., Methods: First, we evaluated the metabolic stability and passive permeability of non-radioactive AZD1283 in vitro. Next, we radiolabeled [ 11 C]AZD1283 with radioactive precursor [ 11 C]NH 4 CN and determined stability in formulation and human plasma. Finally, we investigated the in vivo stability and kinetics of [ 11 C]AZD1283 via dynamic PET imaging of naïve wild-type mice, P2Y12R knockout mouse, and a rhesus macaque., Results: We determined the half-life of AZD1283 in mouse and human liver microsomes to be 37 and > 160 min, respectively, and predicted passive CNS uptake with a small amount of active efflux, using a Caco-2 assay. Our radiolabeling efforts afforded [ 11 C]AZD1283 in an activity of 12.69 ± 10.64 mCi with high chemical and radiochemical purity (>99%) and molar activity of 1142.84 ± 504.73 mCi/μmol (average of n = 3). Of note, we found [ 11 C]AZD1283 to be highly stable in vitro, with >99% intact tracer present after 90 min of incubation in formulation and 60 min of incubation in human serum. PET imaging revealed negligible brain signal in healthy wild-type mice (n = 3) and a P2Y12 knockout mouse (0.55 ± 0.37%ID/g at 5 min post injection). Strikingly, high signal was detected in the liver of all mice within the first 20 min of administration (peak uptake = 58.28 ± 18.75%ID/g at 5 min post injection) and persisted for the remaining duration of the scan. Ex vivo gamma counting of mouse tissues at 60 min post-injection mirrored in vivo data with a mean %ID/g of 0.9% ± 0.40, 0.02% ± 0.01, and 106 ± 29.70% in the blood, brain, and liver, respectively (n = 4). High performance liquid chromatography (HPLC) analysis of murine blood and liver metabolite samples revealed a single radioactive peak (relative area under peak: 100%), representing intact tracer. Finally, PET imaging of a rhesus macaque also revealed negligible CNS uptake/binding in monkey brain (peak uptake = 0.37 Standard Uptake Values (SUV))., Conclusion: Despite our initial encouraging liver microsome and Caco-2 monolayer data, in addition to the observed high stability of [ 11 C]AZD1283 in formulation and human serum, in vivo brain uptake was negligible and rapid accumulation was observed in the liver of both naïve wildtype and P2Y12R knockout mice. Liver signal appeared to be independent of both metabolism and P2Y12R expression due to the confirmation of intact tracer in this tissue for both wildtype and P2Y12R knockout mice. In Rhesus Macaque, negligible uptake of [ 11 C]AZD1283 brain indicates a lack of potential for translation or its further investigation in vivo. P2Y12R is an extremely promising potential PET biomarker, and the data presented here suggests encouraging metabolic stability for this scaffold; however, the mechanism of liver uptake in mice should be elucidated prior to further analogue development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) is associated with aberrant innate immune responses, including microglial activation and infiltration of peripheral myeloid cells into the central nervous system (CNS). Methods to investigate innate immune activation in PD are limited and have not yet elucidated key interactions between neuroinflammation and peripheral inflammation. Translocator protein 18 kDa (TSPO) PET is a widely evaluated imaging approach for studying activated microglia and peripheral myeloid lineage cells in vivo but has yet to be fully explored in PD. Here, we investigate the utility of TSPO PET in addition to PET imaging of triggering receptor expressed on myeloid cells 1 (TREM1)-a novel biomarker of proinflammatory innate immune cells-for detecting innate immune responses in the 6-hydroxydopamine mouse model of dopaminergic neuron degeneration. Methods: C57/BL6J and TREM1 knockout mice were stereotactically injected with 6-hydroxydopamine in the left striatum; control mice were injected with saline. At day 7 or 14 after surgery, mice were administered 18 F-GE-180, 64 Cu-TREM1 monoclonal antibody (mAb), or 64 Cu-isotype control mAb and imaged by PET/CT. Ex vivo autoradiography was performed to obtain high-resolution images of tracer binding within the brain. Immunohistochemistry was conducted to verify myeloid cell activation and dopaminergic cell death, and quantitative polymerase chain reaction and flow cytometry were completed to assess levels of target in the brain. Results: PET/CT images of both tracers showed elevated signal within the striatum of 6-hydroxydopamine-injected mice compared with those injected with saline. Autoradiography afforded higher-resolution brain images and revealed significant TSPO and TREM1 tracer binding within the ipsilateral striatum of 6-hydroxydopamine mice compared with saline mice at both 7 and 14 d after toxin. Interestingly, 18 F-GE-180 enabled detection of inflammation in the brain and peripheral tissues (blood and spleen) of 6-hydroxydopamine mice, whereas 64 Cu-TREM1 mAb appeared to be more sensitive and specific for detecting neuroinflammation, in particular infiltrating myeloid cells, in these mice, as demonstrated by flow cytometry findings and higher tracer binding signal-to-background ratios in brain. Conclusion: TSPO and TREM1 PET tracers are promising tools for investigating different cell types involved in innate immune activation in the context of dopaminergic neurodegeneration, thus warranting further investigation in other PD rodent models and human postmortem tissue to assess their clinical potential., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

Positron emission tomography (PET) is a highly sensitive and versatile molecular imaging modality that leverages radiolabeled molecules, known as radiotracers, to interrogate biochemical processes such as metabolism, enzymatic activity, and receptor expression. The ability to probe specific molecular and cellular events longitudinally in a noninvasive manner makes PET imaging a particularly powerful technique for studying the central nervous system (CNS) in both health and disease. Unfortunately, developing and translating a single CNS PET tracer for clinical use is typically an extremely resource-intensive endeavor, often requiring synthesis and evaluation of numerous candidate molecules. While existing in vitro methods are beginning to address the challenge of derisking molecules prior to costly in vivo PET studies, most require a significant investment of resources and possess substantial limitations. In the context of CNS drug development, significant time and resources have been invested into the development and optimization of computational methods, particularly involving machine learning, to streamline the design of better CNS therapeutics. However, analogous efforts developed and validated for CNS radiotracer design are conspicuously limited. In this Perspective, we overview the requirements and challenges of CNS PET tracer design, survey the most promising computational methods for in silico CNS drug design, and bridge these two areas by discussing the potential applications and impact of computational design tools in CNS radiotracer design.

Background: In the US, EU and elsewhere, basic clinical research studies with positron emission tomography (PET) radiotracers that are generally recognized as safe and effective (GRASE) can often be conducted under institutional approval. For example, in the United States, such research is conducted under the oversight of a Radioactive Drug Research Committee (RDRC) as long as certain requirements are met. Firstly, the research must be for basic science and cannot be intended for immediate therapeutic or diagnostic purposes, or to determine the safety and effectiveness of the PET radiotracer. Secondly, the PET radiotracer must be generally recognized as safe and effective. Specifically, the mass dose to be administered must not cause any clinically detectable pharmacological effect in humans, and the radiation dose to be administered must be the smallest dose practical to perform the study and not exceed regulatory dose limits within a 1-year period. In our experience, the main barrier to using a PET radiotracer under RDRC approval is accessing the required information about mass and radioactive dosing., Results: The University of Michigan (UM) has a long history of using PET radiotracers in clinical research studies. Herein we provide dosing information for 55 radiotracers that will enable other PET Centers to use them under the approval of their own RDRC committees., Conclusions: The data provided herein will streamline future RDRC approval, and facilitate further basic science investigation of 55 PET radiotracers that target functionally relevant biomarkers in high impact disease states.

Background: B cells play a central role in multiple sclerosis (MS) through production of injurious antibodies, secretion of pro-inflammatory cytokines, and antigen presentation. The therapeutic success of monoclonal antibodies (mAbs) targeting B cells in some but not all individuals suffering from MS highlights the need for a method to stratify patients and monitor response to treatments in real-time. Herein, we describe the development of the first CD19 positron emission tomography (PET) tracer, and its evaluation in a rodent model of MS, experimental autoimmune encephalomyelitis (EAE)., Methods: Female C57BL/6 J mice were induced with EAE through immunization with myelin oligodendrocyte glycoprotein (MOG 1-125 ). PET imaging of naïve and EAE mice was performed 19 h after administration of [ 64 Cu]CD19-mAb. Thereafter, radioactivity in organs of interest was determined by gamma counting, followed by ex vivo autoradiography of central nervous system (CNS) tissues. Anti-CD45R (B220) immunostaining of brain tissue from EAE and naïve mice was also conducted., Results: Radiolabelling of DOTA-conjugated CD19-mAb with 64 Cu was achieved with a radiochemical purity of 99% and molar activity of 2 GBq/μmol. Quantitation of CD19 PET images revealed significantly higher tracer binding in whole brain of EAE compared to naïve mice (2.02 ± 0.092 vs. 1.68 ± 0.06 percentage of injected dose per gram, % ID/g, p = 0.0173). PET findings were confirmed by ex vivo gamma counting of perfused brain tissue (0.22 ± 0.020 vs. 0.12 ± 0.003 % ID/g, p = 0.0010). Moreover, ex vivo autoradiography of brain sections corresponded with PET imaging results and the spatial distribution of B cells observed in B220 immunohistochemistry-providing further evidence that [ 64 Cu]CD19-mAb enables visualization of B cell infiltration into the CNS of EAE mice., Conclusion: CD19-PET imaging can be used to detect elevated levels of B cells in the CNS of EAE mice, and has the potential to impact the way we study, monitor, and treat clinical MS.

Neuroinflammation is a key pathologic hallmark of numerous neurologic diseases, however, its exact role in vivo is yet to be fully understood. PET imaging enables investigation, quantification, and tracking of different neuroinflammation biomarkers in living subjects longitudinally. One such biomarker that has been imaged extensively using PET is translocator protein 18 kDa (TSPO). Although imaging TSPO has yielded valuable clinical data linking neuroinflammation to various neurodegenerative diseases, considerable limitations of TSPO PET have prompted identification of other more cell-specific and functionally relevant biomarkers. This review analyzes the clinical potential of available and emerging PET biomarkers of innate and adaptive immune responses, with mention of exciting future directions for the field., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

This communication reports a method for the vinylogous radiofluorination of α-diazoacetates to generate γ-[ 18 F]fluoro-α,β-unsaturated esters and ketones in moderate to good radiochemical yields. The method uses no-carrier-added [ 18 F]AgF and is compatible with aromatic and non-aromatic substrates and a number of different functional groups. The labeling method is showcased in the synthesis of a fluorinated 5-cholesten-3-one derivative as well as a difluorinated product pertinent to drug discovery.

Background: We recently upgraded our [ 18 F]fludeoxyglucose (FDG) production capabilities with the goal of futureproofing our FDG clinical supply, expanding the number of batches of FDG we can manufacture each day, and improving patient throughput in our nuclear medicine clinic. In this paper we report upgrade of the synthesis modules to the GE FASTLab 2 platform (Phase 1) and cyclotron updates (Phase 2) from both practical and regulatory perspectives. We summarize our experience manufacturing FDG on the FASTLab 2 module with a high-yielding self-shielded niobium (Nb) fluorine-18 target., Results: Following installation of Nb targets for production of fluorine-18, a 55 μA beam for 22 min generated 1330 ± 153 mCi of [ 18 F]fluoride. Using these cyclotron beam parameters in combination with the FASTLab 2, activity yields (AY) of FDG were 957 ± 102 mCi at EOS, corresponding to 72% non-corrected AY ( n  = 235). Our workflow, inventory management and regulatory compliance have been greatly simplified following the synthesis module and cyclotron upgrades, and patient wait times for FDG PET have been cut in half at our nuclear medicine clinic., Conclusions: The combination of FASTlab 2 and self-shielded Nb fluorine-18 targets have improved our yield of FDG, and enabled reliable and repeatable manufacture of the radiotracer for clinical use., Competing Interests: Not applicable.Not applicable.The author declare that they have no competing interestsSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Radiolabeled peptides are a valuable class of radiotracer that occupies the space between small molecules and large biologics, and are able to exploit the advantages of both classes of compound. To date, radiolabeled peptides have mainly been utilized in oncology, where the same peptide can often be exploited for diagnostic imaging and targeted radiotherapy by simply varying the radionuclide. In this review, we introduce the main strategies used for synthesis of radiolabeled peptides, and highlight the state of the art for clinical imaging (and therapy) in oncology using the main classes of radiolabeled peptides that have been translated to date. Beyond oncology, radiolabeled peptides are also increasingly being used in other PET applications such as diabetes and cardiac imaging, and we review progress for the new applications., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Review Question/objective: The objective of this review is to identify the effectiveness of storytelling interventions on psychosocial outcomes. In this review, storytelling is where adult patients with a life-threatening illness tell their illness story, facilitated by a healthcare professional.Specifically the review questions are.

Three new positron emission tomography (PET) radiotracers of interest to our functional neuroimaging and translational oncology programs have been prepared through new developments in [(11)C]CO2 fixation chemistry. [(11)C]QZ (glutaminyl cyclase) was prepared via a tandem trapping of [(11)C]CO2/intramolecular cyclization; [(11)C]tideglusib (glycogen synthase kinase-3) was synthesized through a tandem trapping of [(11)C]CO2 followed by an intermolecular cycloaddition between a [(11)C]isocyanate and an isothiocyanate to form the 1,2,4-thiadiazolidine-3,5-dione core; [(11)C]ibrutinib (Bruton's tyrosine kinase) was synthesized through a HATU peptide coupling of an amino precursor with [(11)C]acrylic acid (generated from [(11)C]CO2 fixation with vinylmagnesium bromide). All radiochemical syntheses are fully automated on commercial radiochemical synthesis modules and provide radiotracers in 1-5% radiochemical yield (noncorrected, based upon [(11)C]CO2). All three radiotracers have advanced to rodent imaging studies and preliminary PET imaging results are also reported.

The most commonly utilized PET radionuclide is fluorine-18 ((18)F) because of its convenient half-life and excellent imaging properties. In this review, we present the first analysis of patents issued for radiotracers labeled with fluorine-18 (between 2009 and 2015), and provide perspective on current trends and future directions in PET radiotracer development.

The phenol of 1-(3-(1H-imidazol-1-yl)propyl)-3-(4-hydroxy-3-methoxyphenyl)thiourea was selectively carbon-11 labelled to generate [ 11 C]PBD150 in 7.3% yield from [ 11 C]methyl triflate (non-decay corrected; radiochemical purity ≥95%, specific activity = 5.7 Ci/µmol, n=5). Evaluation of [ 11 C]PBD150 by small animal PET imaging (mouse and rat) determined it does not permeate the blood brain barrier, indicating previously described therapeutic effect in transgenic mice was likely not the result of inhibiting central nervous system glutaminyl cyclase.

Purpose: The National Institutes of Health, American Medical Association, and United States Department of Health and Human Services recommend that patient education materials be written at a fourth to sixth grade reading level to facilitate comprehension. We examined and compared the readability and difficulty of online patient education materials from the American Urological Association and academic urology departments in the Northeastern United States., Materials and Methods: We assessed the online patient education materials for difficulty level with 10 commonly used readability assessment tools, including the Flesch Reading Ease Score, Flesch-Kincaid Grade Level, Simple Measure of Gobbledygook, Gunning Frequency of Gobbledygook, New Dale-Chall Test, Coleman-Liau index, New Fog Count, Raygor Readability Estimate, FORCAST test and Fry score., Results: Most patient education materials on the websites of these programs were written at or above the eleventh grade reading level., Conclusions: Urological online patient education materials are written above the recommended reading level. They may need to be simplified to facilitate better patient understanding of urological topics., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Hyperglycemia in thyrotropin-releasing hormone (TRH) null mice indicates that TRH is involved in the regulation of glucose homeostasis. Further, TRH levels in the pancreas peak during the stages of late embryonic and early neonatal beta cell development. These observations are consistent in linking TRH to islet cell proliferation and differentiation. In this study, we examined the effect of TRH administration in damaged pancreatic rat (streptozotocin, STZ) to determine whether TRH could improve damaged pancreatic beta cells function. We hypothesize that TRH is able to reverse STZ-induced hyperglycemia by increasing pancreatic islet insulin content, preventing apoptosis, and potentially induce islet regeneration. It was found that following intra-peritoneal (ip) injection, TRH (10 microg/kg body weight (bwt)) reverses STZ (65 mg/kg bwt)-induced hyperglycemia (TRH given 3 days after STZ injection). Increased circulating insulin levels and insulin content in extracted pancreas suggests that TRH reversed STZ-induced hyperglycemia through improving pancreatic islet beta cell function. Further studies show a significantly lower level of apoptosis in islets treated with TRH as well as the presence of proliferation marker nestin and Brdu, suggesting that the TRH has the potential to prevent apoptosis and stimulate islet proliferation.

Alteration in the lysosomal system (LS) may represent a central mechanism in neurodegeneration. 6-Hydroxydopamine (6-OHDA) induces oxidative stress and cell death in catecholaminergic cells. The LS and caspases participate in apoptosis, although the mechanism(s) that is involved is not completely understood. Here, we show that Pheochromocytoma (PC12) cells exposed to 6-OHDA results in lysosomal dysregulation, caspase activation and cell death. Cells exposed to 6-OHDA increased expression and release of cystatin C (CC) and suppressed cathepsin B (CB). CB activity significantly declined 24h following exposure to 6-OHDA, however neutralization of CC restored CB activity. Cathepsin D (CD) and caspase-3 activity also increased following exposure to 6-OHDA. Inhibition of CD and caspase-3 with pepstatin A (PA) and DEVD-Cho, respectively, attenuated the 6-OHDA induced cell death at 48 and 72 h. However, the CB inhibitor CA-074 Me failed to protect cells. Additionally, poly-ADP-ribose polymerase (PARP) cleavage was evaluated after exposure to 6-OHDA and PA, CA-074 Me, and DEVD-Cho. Only DEVD-Cho significantly decreased PARP cleavage following exposure to 6-OHDA. Hence, caspase-3 mediated PARP cleavage following exposure to 6-OHDA appears independent of CB and CD alterations. These studies suggest alternate pathways and potential therapeutic targets of cell death associated with oxidative stress, CC, and lysosomal dysregulation.

6-Hydroxydopamine (6-OHDA) is a selective neurotoxin used to induce apoptosis in catecholamine-containing neurons. Although biochemical products and reactive oxygen species (ROS) of 6-OHDA have been well documented, the activation of cellular pathways following exposure are not well understood. Apoptosis in PC12 (Pheochromocytoma) cells was induced by 6-OHDA in a dose (10-150 microM) and time-dependent (24-72 h) manner compared to experimental controls (no treatment). PC 12 cells exposed to 50 microM 6-OHDA demonstrated the involvement of caspase 3 and lysosomal protease alterations. Following 6-OHDA exposure, the caspase 3-like inhibitor Ac-DEVD-CHO significantly decreased 6-OHDA induced cell death. In addition, alterations in expression of the lysosomal cysteine and aspartic proteases, cathepsin B (CB) and cathepsin D (CD) and the endogenous cysteine protease inhibitor cystatin C were observed utilizing immunocytochemical analysis at 24, 48, and 72 h following 6-OHDA exposure. Furthermore, CB and CD and cystatin C immuno-like reactivity was more pronounced in TUNEL positive cells. Moreover, Western blot analysis confirmed a significant increase in protein expression for CB and CD at 72 h and a temporal and concentration dependent increase in cystatin C in response to 6-OHDA. Cells treated with pepstatin A, an inhibitor for CD, showed a significant decrease in cell death, however, CA-074ME, a specific inhibitor for CB, failed to protect cells from 6-OHDA induced cell death. Thus, these results suggest that apoptosis induced by 6-OHDA exposure is mediated in part through caspase 3 activation and lysosomal protease CD.

CD80 and CD86 are important in the initiation of T cell immunity. Although their costimulatory function has long been appreciated, it remains unclear whether the biological significance of the two B7 isoforms resides in their different patterns and kinetics of expression or whether differences exist in their function. We have addressed this issue using HLA-DR1 transfectants co-expressing CD80, CD86, or both molecules as stimulators for naïve, memory, and activated human CD4+ T cells. Both CD80 and CD86 efficiently costimulated alloresponses by unseparated peripheral blood CD4+ T cells; however, CD86 was substantially inferior in costimulating alloresponses by separated memory T cells, and completely incompetent in costimulating three human T cell clones. Furthermore, CD80/CD86 double transfectants stimulated lower responses by the clones than cells expressing CD80 alone. That CD86 was actively inhibitory rather than merely neutral was evidenced by the increase in response to the double CD80/CD86 APC when anti-CD86 antibody was added. Furthermore, addition of anti-CTLA-4 Fab to cultures of HLA-DR1 transfectants co-expressing CD86, fully restored the proliferative response. These results indicate that CD80 and CD86 mediate distinct signals in previously activated T cells, and demonstrate that CTLA-4 ligation may dominate the outcome of CD86-mediated costimulation of activated CD4+ T cells.

Objective: To investigate whether variations within normal ranges of thyroid functioning are related to cognitive and neuropsychiatric functioning in Alzheimer disease (AD)., Background: Mild alterations of thyroid hormone levels, even in the normal range, are associated with changes in mood and cognitive functioning in older, nondemented adults, and lower concentrations of thyroid hormones have been shown to be associated with an increased risk for cognitive decline. Less is known about the relationship between thyroid hormone levels and cognitive and neuropsychiatric dysfunction in AD., Method: Twenty-eight euthyroid patients with AD on donepezil underwent evaluation of thyroid status, including measures of thyroid-stimulating hormone (TSH) and free thyroxine (FT4), and cognitive and neuropsychiatric assessment with the Alzheimer's Disease Assessment Scale, Neuropsychiatric Inventory, and Visual Analog Mood Scales., Results: Correlational analyses indicated statistically significant associations between FT4 concentrations and self-reported feelings of fear and fatigue. Fear and fatigue were negatively correlated with FT4. There were no significant relationships between thyroid hormones and cognition and other depressive and anxiety symptoms., Conclusions: Results of this preliminary study support a relationship between thyroid status and neuropsychiatric symptoms in euthyroid individuals with AD, with lower concentrations of FT4 associated with fear and fatigue.

Background: The response of human CD4+ T cells against porcine cells is of comparable magnitude to that induced by human leukocyte antigen-mismatched allogeneic cells. This reflects productive interactions between key costimulatory molecules across the species barrier. Inhibition of these molecular interactions will be crucial in overcoming CD4+ T-cell-mediated rejection of xenografts. We have performed a detailed investigation to determine the expression profiles and relative contributions of the three key costimulatory molecules in the porcine-human xenogeneic response. Whereas only porcine CD86 is constitutively expressed on resting endothelial cells, both CD40 and CD80 are rapidly expressed after activation. All three costimulatory molecules are expressed by professional antigen-presenting cells., Methods: We have isolated full-length cDNA clones for human and porcine CD80, CD86, and CD40. Human fibroblast cell lines (M1) coexpressing DR1 were transfected with these cDNAs and used in mixed lymphocyte reactions and flow cytometric studies in vitro., Results: These data provide the first characterization of the expression profile and functional role of porcine CD80. Functional assays demonstrate that pCD40, pCD80, and pCD86 are independently capable of costimulating human CD4+ T cells, albeit with differing kinetics. Proliferative responses were of comparable magnitude to those obtained when costimulation was provided by human CD40, CD80, and CD86., Conclusions: These data have implications for therapy targeting the direct pathway of xenorecognition; costimulatory molecule blockade must be directed against both the B7/CD28 and CD40/CD40L pathways.

Optimal proliferation of T cells, although initiated via ligation of the CD3/TCR complex, requires additional costimulatory signals. While the most well-defined in vitro pathway of costimulation is via the B7 family of molecules, a large body of data clearly demonstrates an in vivo role for the CD40/CD154 pathway in transplantation, autoimmunity and allergy. We have examined the role of CD40 as an independent costimulatory molecule by generating a panel of transfected human fibroblasts expressing DR1 with either CD80, CD86 or CD40. Functional assays using allogeneic CD4(+) T cells as responders demonstrated that CD40 was capable of costimulating CD4(+) T cell proliferation particularly in the CD45RO subset. Costimulation by CD40 induced much higher levels of IL-10 than were induced by B7-expressing cells. On day 3 the dominant costimulation was provided by CD40, while by day 5 this was overshadowed by CD80 and CD86. Nonetheless, the provision of costimulation by CD40 was enough to expand an alloreactive T cell line. These results were confirmed in experiments using primary cultures of CD40(+)CD80/CD86(-) renal tubular epithelial cells as the stimulator population. Thus, CD40 is capable of functioning independently (in the absence of B7) as a costimulatory molecule both in terms of proliferation and cytokine release. The data have interesting implications concerning the consequences of antigen presentation by tissue parenchymal cells.

Thyrotropin-releasing hormone (TRH) is best known for its hypothalamic neuroendocrine role in regulating thyroid function. In extra-hypothalamic regions in vitro, we have shown TRH to have a protective effect against synaptic loss and neuronal apoptosis. A role for TRH in Alzheimer's disease (AD) has not been established previously. In this study, we examined the content of the TRH peptide in the hippocampus of elderly controls (n=5) and AD patients (n=7) by radioimmunoassay (RIA). The TRH concentration was decreased in the AD hippocampus compared to normal elderly controls (p < 0.01). In a separate series of experiments utilizing primary cell cultures made from rat hippocampus, TRH peptide concentration was depleted by the addition of TRH antiserum. TRH withdrawal was found to enhance the activity of glycogen synthetase kinase-3 (GSK-3beta), a critical enzyme necessary for the phosphorylation of tau, as well as the phosphorylation of the tau protein itself. This TRH depletion induced upregulation in phosphorylation that was observed to initiate axonal retraction in cultured neurons. These data suggest that TRH within the hippocampus can regulate the activity of various proteins by phosphorylation/dephosphorylation that may be involved in the pathogenesis of AD.

Objective: The purpose of this study was to evaluate the response of pituitary adenomas to radiosurgery as manifested by changes in size and appearance on serial MR imaging., Materials and Methods: Over a mean follow-up period of 36 months, changes in 44 pituitary adenomas were assessed on 147 enhanced MR imaging studies. Prior surgery had been performed in 36 tumors (82%)., Results: At the time of radiosurgery, mean tumor volume was 5.9 +/- 0.8 cm(3) (mean diameter, 2.2 cm). The mean reduction in volume at last follow-up was 41% (+/- 5%, p < 0.001), and a decrease in tumor volume of 25-100% was observed in 34 tumors (77%). Mean reduction in tumor volume at 6 months after radiosurgery was 9% (p = 0.095); at 1 year, 24% (p < 0.001); at 2 years, 34% (p < 0.001); at 3 years, 41% (p < 0.001); and at 4 years, 50% (p = 0.008). Six months after radiosurgery a slight and transient increase in size was observed in 21% of tumors. During follow-up, neither decreased contrast enhancement nor cyst development was associated with changes in tumor volume., Conclusion: Tumor control was observed for most pituitary adenomas after radiosurgery and occurred gradually over a period of several years. A small increase in tumor size might be observed in the first 6 months after radiosurgery. In most cases, reductions in tumor size were not accompanied by a change in contrast enhancement or cyst formation.

To investigate a possible neurotropic role for thyrotropin-releasing hormone (TRH) in the central nervous system, we used recombinant antisense TRH adenovirus (TRHav) to "knock out" TRH in cultured 17-d fetal rat diencephalon. The morphology along with beta-galactosidase (beta-gal) enzyme histochemistry (X-gal staining) and TRH content (femtomoles/well) were used to measure the effect of antisense TRH virus. Control adenovirus mediated beta-gal transfection efficiency was nearly 85%, as shown by positive X-gal staining, and was without effect on cell morphology, TRH content, or the normal response to glucocorticoid (dexamethasone) exposure with enhanced TRH expression. A significant 90% decline in TRH content as well as changes in neuronal morphology (shrunken cell bodies and short dendrites) were observed after 14 but not 7 d following TRHav treatment. The addition of synthetic TRH peptide at 2.5 microM along with TRHav, but not dexamethasone, partly prevented the morphologic changes. No morphologic changes were seen in wild-type AtT20 cells, a pituitary cell line that does not produce TRH. To investigate whether neuronal death from loss of proTRH was owing to apoptosis, neuronal DNA change by means of fluorescent dye H-33342 staining, TUNEL staining, and DNA laddering analysis was examined. Eighty to 90% positive H-33342 and TUNEL staining as well as a 180- to 200-bp DNA fragment on DNA laddering analysis were found as compared to control. These results indicate that proTRH gene expression prevents neuronal apoptosis and may play a role in neuronal development and function.

Because cocaine- and amphetamine-regulated transcript (CART) coexists with alpha-melanocyte stimulating hormone (alpha-MSH) in the arcuate nucleus neurons and we have recently demonstrated that alpha-MSH innervates TRH-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN), we raised the possibility that CART may also be contained in fibers that innervate hypophysiotropic thyrotropin-releasing hormone (TRH) neurons and modulate TRH gene expression. Triple-labeling fluorescent in situ hybridization and immunofluorescence were performed to reveal the morphological relationships between pro-TRH mRNA-containing neurons and CART- and alpha-MSH-immunoreactive (IR) axons. CART-IR axons densely innervated the majority of pro-TRH mRNA-containing neurons in all parvocellular subdivisions of the PVN and established asymmetric synaptic specializations with pro-TRH neurons. However, whereas all alpha-MSH-IR axons in the PVN contained CART-IR, only a portion of CART-IR axons in contact with pro-TRH neurons were immunoreactive for alpha-MSH. In the medial and periventricular parvocellular subdivisions of the PVN, CART was co-contained in approximately 80% of pro-TRH neuronal perikarya, whereas colocalization with pro-TRH was found in <10% of the anterior parvocellular subdivision neurons. In addition, >80% of TRH/CART neurons in the periventricular and medial parvocellular subdivisions accumulated Fluoro-Gold after systemic administration, suggesting that CART may serve as a marker for hypophysiotropic TRH neurons. CART prevented fasting-induced suppression of pro-TRH in the PVN when administered intracerebroventricularly and increased the content of TRH in hypothalamic cell cultures. These studies establish an anatomical association between CART and pro-TRH-producing neurons in the PVN and demonstrate that CART has a stimulatory effect on hypophysiotropic TRH neurons by increasing pro-TRH gene expression and the biosynthesis of TRH.

The CTLA4 receptor (CD152) on activated T lymphocytes binds B7 molecules (CD80 and CD86) on APC and delivers a signal that inhibits T cell proliferation. Several regions involved in binding to B7 are known, but the relative importance of these is not clear. We have cloned porcine CTLA4 (pCTLA4). Although highly homologous to human CTLA4 (hCTLA4), the predicted protein sequence contains a leucine for methionine substitution at position 97 in the MYPPPY sequence. A fusion protein constructed from the extracellular regions of pCTLA4 and the constant regions of human IgG1 (pCTLA4-Ig) bound porcine CD86 with equivalent affinity to that of hCTLA4-Ig. However, pCTLA4-Ig bound poorly to human CD80 and CD86 expressed on transfectants and EBV-transformed human B cells. In functional assays with MHC class II-expressing porcine endothelial cells and human B cells, pCTLA4-Ig blocked human CD4+ T cell responses to pig but not human cells, whereas control hCTLA4-Ig inhibited responses to both. Comparison between mouse, human, and porcine CTLA4-Ig suggests that the selective binding of pCTLA4-Ig to porcine CD86 molecules is due to the L for M substitution at position 97. Our results indicate that pCTLA4-Ig may be a useful reagent to define the precise nature of the interaction between B7 and CTLA4. By failing to inhibit the delivery of costimulatory signals provided by human B7, it may also prove to be a relatively specific inhibitor of the direct human T cell response to immunogenic pig tissue.

Stereotactic radiosurgery with the gamma knife delivers focused radiation from a cobalt-60 source in a single session to a pituitary tumour with minimal radiation to the adjacent normal brain tissue. Currently, gamma knife radiosurgery is predominantly used to treat failed pituitary surgery, although it has a role as a primary treatment for patients unwilling or unsuitable, for medical reasons, to undergo trans-sphenoidal surgery. The major risk from gamma knife radiosurgery is radiation damage to the visual pathways, but this can be avoided by limiting the radiation dose to the optic chiasm to under 10 Gy. In contrast, the neuronal and vascular structures running in the cavernous sinus are much less radiosensitive, allowing an ablative dose to be administered to tumours showing lateral invasion and impinging on cranial nerves III, IV, V and VI. Gamma knife radiosurgery appears to produce remission in secretory tumours faster than fractionated radiotherapy. Furthermore, the potential long-term risk of developing a second extra-pituitary brain tumour, as well as the neuropsychiatric effects associated with conventional radiation administration, seems less likely to occur with this form of treatment.

To determine whether c-fos/c-jun (AP-1) and CREB mediate glucocorticoid stimulated TRH gene regulation, we investigated the effect of N-ethyl-maleimide (NEM), an alkylating agent, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on this process. NEM decreased while TPA increased TRH levels in rat hypothalamic culture, changes similar to their effects on CREB and Fos/Jun proteins in the AtT20 cell line. This suggests that glucocorticoid stimulation of TRH gene expression may be regulated by the AP-1 complex and CREB pathway.

In order to study the effect of glucocorticoids on thyrotropin-releasing hormone (TRH) and protooncogenes, we describe a double labeled in situ hybridization method to explore this issue. The development of non-isotopic in situ hybridization histochemistry has proven to be an important tool for cellular and molecular studies in neurobiology [C.L.E. Moine, E. Normand, B. Bloch, Use of non-radioactive probes for mRNA detection by in situ hybridization: interests and applications in the central nervous system, Cell. Mol. Biol. 41 (1995) 917-923]. These methods involve the anatomic localization of labeled RNA or DNA molecules which hybridize with complementary target RNA or DNA sequences in the cell. With regard to gene expression, in situ hybridization allows the study of specific mRNA levels and the distribution between various cell types. It also allows the comparison of mRNA levels at various stages of development. Double labeled in situ hybridization is able to detect the colocalization of two different mRNAs simultaneously. Accordingly, this approach is utilized for specific studies involving the expression and distribution of TRH mRNA and the protooncogenes, c-fos/c-jun, in cultured rat hypothalamic neurons [L. G. Luo, I.M.D. Jackson, Glucocorticoids stimulate TRH and c-fos/c-jun gene co-expression in cultured hypothalamic neurons, Brain Research 791 (1998) 56-62]. Our protocol for double labeled in situ hybridization reflects a modification of a number of original protocols developed by others [H. Breitschopf, G. Suchanek, R.M. Gould, D.R. Colman, H. Lassmann, In situ hybridization with digoxigenin-labeled probes: sensitive and reliable detection method applied to myelinating rat brain, Acta Neurropathol. 84 (1992) 581-587; S. McQuaid, J. McMahon, G.M. Allan, A comparison of digoxigenin and biotin labeled DNA and RNA probes for in situ hybridization, Biotech. Histochem. 70 (1995) 147-154; E. Hrabovszky, M.E. Vrontakis, S.L. Petersen, Triple-labeling method combining immunocytochemistry and in situ hybridization histochemistry: demonstration of overlap between Fos-immunoreactive and galanin mRNA-expressing subpopulations of luteinizing hormone-releasing hormone neurons in female rats, J. Histochem. Cytochem. 43 (1995) 363-370]. This technique can be readily applied to various studies of cellular gene expression in the mammalian nervous system involving other neural peptides and transcription factors., (Copyright 1999 Elsevier Science B.V.)

Stereotactic radiosurgery with the Gamma Knife allows the delivery of focused radiation in a single session from a Cobalt-60 source to a pituitary tumor with little radiation to surrounding normal brain tissue. At this time the major role for Gamma Knife radiosurgery in acromegaly is for the treatment of failed pituitary surgery although it may also by used as primary treatment for patients unwilling or unsuitable, for medical reasons, to undergo transsphenoidal surgery. The major risk from Gamma Knife radiosurgery appears to be radiation damage to the visual pathways, but this can be obviated by limiting the radiation dose to the optic chiasm under 10 Gy. In contrast, the neuronal and vascular structures running in the cavernous sinus are much less radiosensitive allowing an ablative dose to be administered to tumors showing lateral invasion and impinging on cranial nerves III, IV, V and VI. Gamma Knife radiosurgery appears to produce effects in GH secreting tumors faster than with fractionated radiotherapy without the potential long-term risk of developing a second extrapituitary brain tumor as well as the neuropsychiatric effects associated with conventional radiation administration.

1. Gamma knife therapy is an effective method of delivering radiation to pituitary tumors that have failed surgery and may be used as primary treatment in circumstances in which the patient refuses or is unsuitable for a transsphenoidal procedure. 2. Stereotactic radiosurgery with the gamma knife unit is generally administered in a single session unlike fractionated radiotherapy, which is administered four to five times per week over a 6-week period. 3. Preliminary data suggest that resolution of pituitary hypersecretion is faster with gamma knife therapy than with conventional radiotherapy. 4. Because of the nature of the gamma knife therapy and the fact that the radiation dose conforms to the tumor shape, there is a steep fall-off of radiation to surrounding tissue. Accordingly, the radiation dose to extrapituitary brain is substantially less with gamma knife radiosurgery than with conventional radiotherapy. This suggests that the development of second brain tumors and neurocognitive complications, which are significant risks with conventional radiotherapy, is much less likely with gamma knife surgery. 5. Gamma knife radiosurgery can be used to ablate tumors invading the cavernous sinus. 6. Gamma knife radiosurgery is safe as long as the dose of radiation to the optic structures is kept under 10 Gy. 7. Long-term follow-up is required for pituitary tumors treated by gamma knife therapy so as to determine its efficacy as well as its effects on pituitary function and any resultant complications.

Background: The pituitary thyroid axis is frequently effected in human depression possibly due to alteration in hypothalamic thyrotropin releasing hormone (TRH) secretion. Since clinical recovery is associated with normalization of thyroid function, the direct effect of antidepressants on TRH expression in a well established fetal rat hypothalamic neuronal culture system was investigated., Methods: Fetal rat hypothalamic neurons (day 17) in culture were treated with different concentrations of antidepressants with or without glucocorticoids for 7 days following which TRH content was measured by radioimmunoassay (RIA)., Results: The results showed that Imipramine (IMIP), a tricyclic antidepressant (TCA), decreased the TRH content in a dose-dependent manner (from 80.7 +/- 4.9, at 10(-9) mol/L, to 14.1 +/- 0.6, at 10(-5) mol/L, fmol/well; P < 0.05). Desipramine (DESI), another tricyclic antidepressant, also decreased the TRH content (from 63.6 +/- 2.5, at 10(-9) mol/L, to 12.6 +/- 0.4, at 10(-5) mol/L, fmol/well; P < 0.05). Sertraline (SERT) and Fluoxetine (FLUO), serotonin selective reuptake inhibitors (SSRI), also decreased TRH content in a dose dependent manner (from 83.9 +/- 7.9, at 10(-10) mol/L, to 7.6 +/- 0.4, at 10(-5) mol/L, and from 41.66 +/- 2.5, at 10(-8) mol/L, to 17.54 +/- 0.92, at 10(-6) mol/L, fmol/well, respectively; both P < 0.05). We then tested the effect of these antidepressants on the Dex stimulation of TRH content. IMIP, DESIP and FLUO at 10(-6) mol/L reduced the TRH response to glucocorticoid stimulation (36.4 +/- 4.0, 56.6 +/- 2.4, 23.75 +/- 4.0, respectively vs 107 +/- 7.5 fmol/well; P < 0.05)., Conclusion: This raises the possibility that the enhanced thyroid function in depression, which we postulate, may result in part from glucocorticoid stimulation of TRH gene expression, can be reversed by antidepressants through a direct effect on the TRH neuron. However, other mechanisms may need to be invoked in addition since basal TRH content was also reduced.

Hypothyroidism may give rise to frank depression that usually responds to thyroxine therapy. Depressed subjects with subclinical hypothyroidism and/or autoimmune thyroiditis should probably also be treated similarly. Most patients with depression, although generally viewed as chemically euthyroid, have alterations in their thyroid function including slight elevation of the serum thyroxine (T4), blunted thyrotropin (TSH) response to thyrotropin-releasing hormone (TRH) stimulation, and loss of the nocturnal TSH rise. These changes are generally reversed following alleviation of the depression. The role of adjuvant triiodothyronine (T3) treatment in resistant depression has not been established, but the data suggest that it will be beneficial in about 25% of cases. However, controlled trials to establish this approach are needed. The underlying mechanism leading to the beneficial response from T3 is unknown, but may reflect brain hypothyroidism in the context of systemic euthyroidism. The hypothalamus in culture, which is analogous to a deafferentated hypothalamus in vivo, shows a paradoxic increase in TRH production after glucocorticoid stimulation. It is known that in human depression there is a functional disconnection of the hypothalamus with impairment of the inhibitory glucocorticoid feedback pathway from the hippocampus to the hypothalamus that results in the typical elevated cortisol levels and impaired dexamethasone suppression. It is postulated that a similar situation prevails with regards to the thyroid axis and that the increased T4 in depression, as well as the blunted TSH response to exogenous TRH, reflects glucocorticoid activation of the TRH neuron leading to increased TRH secretion with resultant down regulation of the TRH receptor on the thyrotrope. Normalization of thyroid function after treatment may result in part from an inhibitory response of the TRH neuron to antidepressant medication, although other effects may also be responsible.

Disulfiram (Dis), an inhibitor of peptidyl-glycine alpha-amidating monooxygenase, the enzyme responsible for the production of alpha-amidated peptides from their immediate, glycine-extended precursors was used to investigate the paracrine effects of TRH on anterior pituitary (AP) hormone secretion. It reduces the production of TRH without directly affecting the classical pituitary hormones, none of which is amidated. Dis (8 microM) decreased the accumulation of TRH accompanied by an equimolar increase in TRH-Gly levels, indicating that pro-TRH biosynthesis persisted. TRH and TSH release into the medium was significantly lowered, whereas other pituitary hormones were unaffected. In contrast, dexamethasone (10 nM), which up-regulates TRH gene expression in this system, increased TRH (+89.5%) and TSH (+61.3%) secretion. The combination of dexamethasone and Dis further diminished the release of TRH (-73%) and TSH (-40.3%) observed with Dis alone, indicating that TRH synthesized within the AP regulates TSH secretion. Dis significantly elevated prepro-TRH (25-50) and pro-TRH messenger RNA levels, suggesting that reduced TRH formation leads to increased pro-TRH biosynthesis and that TRH regulates its own secretion. Thus, TRH synthesized by cultured AP cells not only stimulates TSH release through a paracrine effect, but has a negative feedback on its own biosynthesis by an autocrine mechanism.

To explore whether the protooncogenes, c-fos/c-jun, might be involved in regulating the effect of glucocorticoids on thyrotropin-releasing hormone in fetal rat diencephalic neurons, their localization and transcriptional activity were investigated using double-labeled in situ hybridization, Northern blot and nuclear run-on assays. The results showed that TRH mRNA was coexpressed with both c-jun and c-fos in the same neurons. Treatment with dexamethasone, a synthetic glucocorticoid, at 10(-8) M, enhanced transcriptional activity resulting in an increase in both cell number and intensity of all three mRNAs. The existence of c-fos/c-jun in thyrotropin-releasing hormone neurons and the increased transcriptional activity following dexamethasone treatment suggests that these protooncogenes could mediate the effect of glucocorticoids on thyrotropin-releasing hormone gene expression., (Copyright 1998 Elsevier Science B.V.)

The proto-oncogenes, cfos/cjun, are co-localized with thyrotropin-releasing hormone (TRH) in cultured anterior pituitary cells and increase following exposure to dexamethasone (Dex). To assess the role of cfos and cjun in the Dex stimulation of TRH gene expression, we used antisense oligonucleotides to block cfos and cjun expression in order to reduce formation of activating protein-1 (AP-1). The results showed that the antisense oligonucleotides together effectively reduced cfos/cjun gene expression and consequently the glucocorticoid stimulation of TRH peptide and mRNA. The findings indicate that cfos/cjun are involved in the glucocorticoid activation of TRH gene expression.

The processing of pro-TRH, has been extensively studied in our laboratory using a corticotropic cell line, AtT20, transfected with the pro-TRH gene. We have also demonstrated that the convertases PC1 and PC2 process pro-TRH to cryptic peptides in vitro. However, although these processing pathways have been well characterized in vitro, little is known about the processing and subcellular distribution of pro-TRH and its derived peptides in hypothalamic neurons, an endogenous source of pro-TRH and PC enzymes. In this study we used multiple approaches to identify, both biochemically and anatomically, the presence and localization of pro-TRH (26 kDa) and its processing products. We also investigated the presence of PC1 and PC2 enzymes and the coexpression of pro-TRH and PC1 messenger RNAs. Identification of the TRH precursor was demonstrated by 1) Western blot analysis of cellular extracts, 2) immunoprecipitation of radiolabeled pro-TRH followed by analysis on acrylamide gel electrophoresis, 3) fluorescence immunocytochemistry, and 4) immunoelectron microscopy. The presence of the convertases PC1 and PC2 was determined by Western blot analysis of cellular extracts and fluorescence immunocytochemistry. The coexpression of pro-TRH with PC1 was shown by double in situ hybridization. Our findings support three main conclusions. First, this primary culture system of hypothalamic neurons is suitable for characterizing pro-TRH processing as well as identifying the anatomical location of its processing products. Second, prohormome processing takes place during axonal transport after removal of the signal peptide in the endoplasmic reticulum, and subsequent cleavages of the prohormone occur as intermediate peptides move down the axon toward the nerve terminal. This coupled transport-processing phenomenon may provide the necessary mechanism to ensure flexibility in differential processing of specific protein sequences that are determined by the secretory needs of cells. It appears that certain intermediate peptides differ in their subcompartmental distribution, suggesting the possibility of a differential processing and maturation of pro-TRH-derived peptides. Thirdly, the 87-kDa form of PC 1 may initiate the processing of pro-TRH at the Golgi complex level, which then continues to be processed by PC1 and PC2 in later stages of the secretory pathway.