Human Pituitary Gland Research Articles

Regulation of immune cells by miR-451 and its potential as a biomarker in immune-related disorders: a mini review.

In 2005, Altuvia and colleagues were the first to identify the gene that encodes miR-451 in the human pituitary gland, located in chromosome region 17q11.2. Subsequent studies have confirmed that miR-451 regulates various immune cells, including T cells, B cells, microglia, macrophages, and neutrophils, thereby influencing disease progression. The range of immune-related diseases affected encompasses various cancers, lymphoblastic leukemia, and injuries to the lungs and spinal cord, among others. Moreover, miR-451 is produced by immune cells and can regulate both their own functions and those of other immune cells, thus creating a regulatory feedback loop. This article aims to comprehensively review the interactions between miR-451 and immune cells, clarify the regulatory roles of miR-451 within the immune system, and assess its potential as both a therapeutic target and a biomarker for immune-related diseases.

Activated AMP-protein kinase (pAMPK) is overexpressed in human somatotroph pituitary adenomas

IntroductionAMPK (AMP-activated protein kinase) is an enzyme that acts as a metabolic sensor and regulates multiple pathways via phosphorylating proteins in metabolic and proliferative pathways. The aim of this work was to study the activated cellular AMPK (phosphorylated-AMPK at Thr172, pAMPK) levels in pituitary tumor samples from patients with sporadic and familial acromegaly, as well as in samples from normal human pituitary gland. MethodsWe studied pituitary adenoma tissue from patients with sporadic somatotroph adenomas, familial acromegaly with heterozygote germline variants in the aryl hydrocarbon receptor interacting protein (AIP) gene (p.Q164*, p.R304* and p.F269_H275dup) and autopsy from normal pituitary glands without structural alterations. ResultsCellular levels of pAMPK were significantly higher in patients with sporadic acromegaly compared to normal pituitary glands (p < 0.0001). Tissues samples from patients with germline AIP mutations also showed higher cellular levels of pAMPK compared to normal pituitary glands. We did not observe a significant difference in cellular levels of pAMPK according to the cytokeratin (CAM5.2) pattern (sparsely or densely granulated) for tumor samples of sporadic acromegaly. ConclusionOur data show, for the first time in human cells, an increase of cellular levels of pAMPK in sporadic somatotropinomas, regardless of cytokeratin pattern, as well as in GH-secreting adenomas from patients with germline AIP mutations.

Flexible in-cavity MRI receiving coil for ultra-high-resolution imaging of the pituitary gland.

The objective of this study was the preclinical design and construction of a flexible intrasphenoid coil aiming for submillimeter resolution of the human pituitary gland. Sphenoid sinus measurements determined coil design constraints for use in > 95% of adult patients. Temperature safety parameters were tested. The 2-cm-diameter coil prototype was positioned in the sphenoid sinus of cadaveric human heads utilizing the transnasal endoscopic approach that is used clinically. Signal-to-noise ratio (SNR) was estimated for the transnasal coil prototype compared with a standard clinical head coil. One cadaveric pituitary gland was explanted and histologically examined for correlation to the imaging findings. With the coil positioned directly atop the sella turcica at a 0° angle of the B0 static field, the craniocaudal distance (21.2 ± 0.8 mm) was the limiting constraint. Phantom experiments showed no detectable change in temperature at two sites over 15 minutes. The flexible coil was placed transnasally in cadaveric specimens using an endoscopic approach. The image quality was subjectively superior at higher spatial resolutions relative to that with the commercial 20-channel head coil. An average 17-fold increase in the SNR was achieved within the pituitary gland. Subtle findings visualized only with the transnasal coil had potential pathological correlation with immunohistochemical findings. A transnasal radiofrequency coil feasibly provides a 17-fold boost in the SNR at 3 T. The ability to safely improve the quality of pituitary imaging may be helpful in the identification and subsequent resection of small functional pituitary lesions.

Pharmacokinetic Analysis of [18F]FES PET in the Human Brain and Pituitary Gland

PurposeEstrogen receptors (ER) are implicated in psychiatric disorders. We assessed if ER availability in the human brain could be quantified using 16α-[18F]-fluoro-17β-estradiol ([18F]FES) positron emission tomography (PET).ProceduresSeven post‑menopausal women underwent a dynamic [18F]FES PET scan with arterial blood sampling. A T1-weighted MRI was acquired for anatomical information. After one week, four subjects received a selective ER degrader (SERD), four hours before the PET scan. Pharmacokinetic analysis was performed using a metabolite-corrected plasma curve as the input function. The optimal kinetic model was selected based on the Akaike information criterion and standard error of estimated parameters. Accuracy of Logan graphical analysis and standardized uptake value (SUV) was determined via correlational analyses.ResultsThe reversible two-tissue compartment model (2T4k) model with fixed K1/k2 was preferred. The total volume of distribution (VT) could be more reliably estimated than the binding potential (BPND). A high correlation of VT with Logan graphical analysis was observed, but only a moderate correlation with SUV. SERD administration resulted in a reduced VT in the pituitary gland, but not in other regions.ConclusionsThe optimal quantification method for [18F]FES was the 2T4k with fixed K1/k2 or Logan graphical analysis, but specific binding was only observed in the pituitary gland.

Сytotoxic T lymphocyte-associated protein 4 (CTLA4) is overexpressed in a subset of prolactin- and growth hormone-secreting pituitary adenomas.

Cytotoxic T lymphocyte-associated protein 4 (CTLA4), a negative regulator typically expressed on the surface of T lymphocytes, is targeted by immunotherapy in patients with an ever-expanding spectrum of cancers. Characterizing the expression of CTLA4 in the pituitary gland could provide additional rationale for using immune checkpoint inhibitors in pituitary adenoma patients who do not respond to conventional treatments. We assessed the expression of CTLA4 mRNA and protein in a panel of 157 human pituitary glands, 45 collected at autopsy and 112 at surgery. These specimens included 50 normal glands and 107 adenomas: 41 nonsecreting, 25 PRL-, 24 ACTH-, 11 GH-, 2 TSH-, 1 FSH-secreting, and 3 atypical. Specimens were stained for CTLA4 and adenohypophyseal hormones using RNAscope in situ hybridization, immunohistochemistry, and RNAscope Multiplex Fluorescent Assay. CTLA4 mRNA was detectable in most normal pituitary glands (48 of 50, 96%) but varied in expression, with a histological score (H-score) ranging from 0.6 to 20. The variation did not depend upon the patient's gender and age and was not significantly affected by the archival storage time. CTLA4 expression was higher (P = 0.022) in pituitary adenomas than normal glands, with the greatest levels seen in PRL- and GH-secreting adenomas (P = 0.009 and 0.023 versus normal, respectively). Eight of 25 (32%) prolactinomas and 3 of 11 (27%) GH-adenomas had an H-score greater than 20, while no differences were seen for the other types. These novel data highlight the expression of an immune checkpoint such as CTLA4 on pituitary endocrine cells, a finding that could be exploited for therapeutical applications.

Development of a bespoke phantom to optimize molecular PET imaging of pituitary tumors

BackgroundImage optimization is a key step in clinical nuclear medicine, and phantoms play an essential role in this process. However, most phantoms do not accurately reflect the complexity of human anatomy, and this presents a particular challenge when imaging endocrine glands to detect small (often subcentimeter) tumors. To address this, we developed a novel phantom for optimization of positron emission tomography (PET) imaging of the human pituitary gland. Using radioactive 3D printing, phantoms were created which mimicked the distribution of 11C-methionine in normal pituitary tissue and in a small tumor embedded in the gland (i.e., with no inactive boundary, thereby reproducing the in vivo situation). In addition, an anatomical phantom, replicating key surrounding structures [based on computed tomography (CT) images from an actual patient], was created using material extrusion 3D printing with specialized filaments that approximated the attenuation properties of bone and soft tissue.ResultsThe phantom enabled us to replicate pituitary glands harboring tumors of varying sizes (2, 4 and 6 mm diameters) and differing radioactive concentrations (2 ×, 5 × and 8 × the normal gland). The anatomical phantom successfully approximated the attenuation properties of surrounding bone and soft tissue. Two iterative reconstruction algorithms [ordered subset expectation maximization (OSEM); Bayesian penalized likelihood (BPL)] with a range of reconstruction parameters (e.g., 3, 5, 7 and 9 OSEM iterations with 24 subsets; BPL regularization parameter (β) from 50 to 1000) were tested. Images were analyzed quantitatively and qualitatively by eight expert readers. Quantitatively, signal was the highest using BPL with β = 50; noise was the lowest using BPL with β = 1000; contrast was the highest using BPL with β = 100. The qualitative review found that accuracy and confidence were the highest when using BPL with β = 400.ConclusionsThe development of a bespoke phantom has allowed the identification of optimal parameters for molecular pituitary imaging: BPL reconstruction with TOF, PSF correction and a β value of 400; in addition, for small (< 4 mm) tumors with low contrast (2:1 or 5:1), sensitivity may be improved using a β value of 100. Together, these findings should increase tumor detection and confidence in reporting scans.

Pituitary adenomas evade apoptosis via noxa deregulation in Cushing's disease.

SUMMARYSporadic pituitary adenomas occur in over 10% of the population. Hormone-secreting adenomas, including those causing Cushing’s disease (CD), cause severe morbidity and early mortality. Mechanistic studies of CD are hindered by a lack of in vitro models and control normal human pituitary glands. Here, we surgically annotate adenomas and adjacent normal glands in 25 of 34 patients. Using single-cell RNA sequencing (RNA-seq) analysis of 27594 cells, we identify CD adenoma transcriptomic signatures compared with adjacent normal cells, with validation by bulk RNA-seq, DNA methylation, qRT-PCR, and immunohistochemistry. CD adenoma cells include a subpopulation of proliferating, terminally differentiated corticotrophs. In CD adenomas, we find recurrent promoter hypomethylation and transcriptional upregulation of PMAIP1 (encoding pro-apoptotic BH3-only bcl-2 protein noxa) but paradoxical noxa downregulation. Using primary CD adenoma cell cultures and a corticotroph-enriched mouse cell line, we find that selective proteasomal inhibition with bortezomib stabilizes noxa and induces apoptosis, indicating its utility as an anti-tumor agent.

The human pituitary master gland stripped to single-cell resolution.

Stem cells in the pituitary master gland remain mystifying; moreover, they have hardly been explored in humans. A new study applied transcriptome and chromatin accessibility profiling at single-cell resolution to human postmortem pituitary glands from individuals of different ages, thereby providing insights into the heterogeneity and regulatory mechanisms of the projected human pituitary stem cell population.

508 Syngeneic Pair-wise Transcriptomic/Epigenomic Analysis of Sporadic Cushing’s Adenomas and Adjacent Gland Reveals Novel Tumorigenic Pathways

INTRODUCTION: Sporadic pituitary adenomas are found in more than 10% of the general population and are mutationally bland. The normal human pituitary transcriptome is unknown, precluding syngeneic studies on Cushing's disease (CD) pathogenesis. METHODS: We surgically annotated adenomas and adjacent normal glands and performed single cell RNAseq (scRNAseq; 10X Genomics Chromium 3’) on 28,724 cells from 6 patients (3 CD and 3 non-CD). We performed bulk RNAseq (Illumina Nextseq) in 7 patients (5 CD and 2 non-CD) and DNA methylation analysis (Illumina Infinium MethylEPIC 850k) in 6 patients (3 CD and 3 non-CD). Transcriptomic/epigenomic data were analyzed using R packages and custom Python pipelines for pairwise differential expression analyses. RESULTS: We mapped the post-natal human pituitary gland at single cell resolution and identified canonical resident pituitary cell classes. Corticotrophs were abundant within CD adenomas but rare in the surrounding normal gland, with downregulated POMC expression consistent with repression in the hypercortisolemic state. CD corticotrophs overexpressed signature genes PCSK1, PPP1R17, EGLN, RSPO3 and PMAIP1 compared to surrounding tissues. This gene signature was specific for core adenomas and correlated with anatomic demarcation at the time of surgery. We confirmed overexpression of PCSK1, PPP1R17, RSPO3 and PMAIP1 by independent RNAseq. We identified hypomethylation at the promoters of PMAIP1, PPP1R17 and RSPO3 but not PCSK1 or EGLN1 in CD adenomas. CONCLUSION: We mapped the transcriptome of the adult human pituitary gland, identified novel signature genes for CD and highlighted DNA methylation as an epigenetic mechanism of transcriptional activation in a number of Cushing’s adenoma signature genes. We provide novel, targetable mechanistic pathways for therapeutic intervention in Cushing’s disease.

The Upregulation of Molecules Related to Tumor Immune Escape in Human Pituitary Adenomas.

Human pituitary adenomas are one of the most common intracranial neoplasms. Although most of these tumors are benign and can be treated medically or by transsphenoidal surgery, a subset of these tumors are fast-growing, aggressive, recur, and remain a therapeutic dilemma. Because antibodies against immune checkpoint receptors PD-1 and CLTA-4 are now routinely used for cancer treatment, we quantified the expression of mRNA coding for PD-1, CLTA-4, and their ligands, PD-L1, PD-L2, CD80, and CD86 in human pituitary adenomas and normal pituitary glands, with the ultimate goal of exploiting immune checkpoint therapy in aggressive pituitary adenomas. Aggressive pituitary adenomas demonstrated an increased expression of PD-L2, CD80, and CD86 in compared to that of normal human pituitary glands. Furthermore, aggressive pituitary tumors demonstrated significantly higher levels of CD80 and CD86 compared to non-aggressive tumors. Our results establish a rationale for studying a potential role for immune checkpoint inhibition therapy in the treatment of pituitary adenomas.

Morpho-Molecular Metabolic Analysis and Classification of Human Pituitary Gland and Adenoma Biopsies Based on Multimodal Optical Imaging.

Simple SummaryThe pituitary gland governs the function of nearly all endocrine glands and pituitary oncogenesis often distorts the hormonal balance. For optimal surgical cure it is crucial to discriminate pathological tissue from intact pituitary gland. Our multimodal imaging approach allows for morpho-molecular metabolic analysis and discrimination of pituitary gland and adenomas combining different complementary techniques such as optical coherence tomography (OCT), multiphoton microscopy (MPM) and line scan Raman microspectroscopy (LSRM). Radiomics as well as analysis of spectroscopic features allows enhanced discrimination of pituitary gland and adenomas and, furthermore, classification of pituitary adenoma subtypes.Pituitary adenomas count among the most common intracranial tumors. During pituitary oncogenesis structural, textural, metabolic and molecular changes occur which can be revealed with our integrated ultrahigh-resolution multimodal imaging approach including optical coherence tomography (OCT), multiphoton microscopy (MPM) and line scan Raman microspectroscopy (LSRM) on an unprecedented cellular level in a label-free manner. We investigated 5 pituitary gland and 25 adenoma biopsies, including lactotroph, null cell, gonadotroph, somatotroph and mammosomatotroph as well as corticotroph. First-level binary classification for discrimination of pituitary gland and adenomas was performed by feature extraction via radiomic analysis on OCT and MPM images and achieved an accuracy of 88%. Second-level multi-class classification was performed based on molecular analysis of the specimen via LSRM to discriminate pituitary adenomas subtypes with accuracies of up to 99%. Chemical compounds such as lipids, proteins, collagen, DNA and carotenoids and their relation could be identified as relevant biomarkers, and their spatial distribution visualized to provide deeper insight into the chemical properties of pituitary adenomas. Thereby, the aim of the current work was to assess a unique label-free and non-invasive multimodal optical imaging platform for pituitary tissue imaging and to perform a multiparametric morpho-molecular metabolic analysis and classification.

Pituitary Adenomas Evade Apoptosis via Noxa Deregulation in Cushing's Disease

Introduction: Sporadic pituitary adenomas are common (>10%) in the general population. Microadenomas (< 1 cm across) can become symptomatic with adrenocorticotropin oversecretion causing Cushing’s disease (CD), leading to high morbidity and mortility. Pathogenic mechanisms in a majority of adenomas including wildtype CD adenomas remain unknown. Mechanistic studies are hindered by a lack of in-vitro models and normal human pituitary gland controls. We hypothesized that comparative analysis of human adenomas with syngeneic normal pituitary glands could reveal pathogenic mechanisms driving CD. Methods: We surgically annotated adenomas and en-route adjacent normal glands in 34 patients. We performed single cell RNAseq and captured 28,724 cells in 6 patients (3 CD and 3 non-CD; 5 pairwise), bulk RNAseq in 7 patients (5 CD and 2 non-CD; 5 pairwise) and DNA methylation analysis in 6 patients (3 CD and 3 non-CD). We confirmed the mechanisms of sporadic CD adenoma pathogenesis in 2 additional syngeneic pairs of human cell lines and using a novel murine normal corticotroph-enriched cell line. Results: Using marker-based classification, we confirmed canonical resident cell classes including corticotrophs in the human pituitary gland. We found SOX2+/S100B+ folliculostellate cells which play a role in pituitary cell replenishment and homeostasis in the normal gland but not in the adenoma compartment. Within the terminally differentiated corticotrophs in the adenoma compartment, we discovered a subset of proliferating cells with upregulation of canonical oncogenes, histone modifiers and DNA repair genes. We also found that consensus dominant genes including PMAIP1 (encoding the pro-apoptotic BH3 only Bcl-2 protein noxa) sharply demarcated adenoma cells from normal gland. The PMAIP1 locus was hypomethylated in CD adenomas compared to non-CD samples. However, despite epigenetic and transcriptional upregulation, we found proteasomal degradation of noxa protein in CD adenomas. Using the selective proteasomal inhibitor bortezomib, we were able to rescue noxa ex-vivo and in-vitro, induce apoptosis, and reduce cell survival of adenoma cell lines. Conclusions: We defined the transcriptionl and DNA methylation landscape of human pituitary adenomas compared to adjacent normal gland tissue. We identified proteasomal-mediated noxa degradation despite PMAIP1 overexpression as an escape mechanism from apoptosis in adenomatous corticotrophs. The proteasomal inhibitor bortezomib rescued noxa and induced apoptosis in adenoma cells, highlighting its potential as a novel therapeutic strategy in CD.

Human pituitary development and application of iPSCs for pituitary disease.

The pituitary plays a pivotal role in maintaining systemic homeostasis by secreting several hormones. During fetal development, the pituitary develops from the oral ectoderm in contact with the adjacent hypothalamus. This process is regulated by the fine-tuned expression of transcription and growth factors. Impairments of this process result in congenital pituitary hypoplasia leading to dysfunction of the pituitary. Although animal models such as knockout mice have helped to clarify these underlying mechanisms, the developmental processes of the human pituitary gland and the mechanisms of human pituitary disorders have not been fully understood. This is because, at least in part, of the lack of a human pituitary developmental model. Recently, methods for in vitro induction of the pituitary gland from human pluripotent stem cells were developed. These models can be utilized not only for regenerative medicine but also for human pituitary studies on developmental biology and for modeling of pituitary disorders, such as hypopituitarism and pituitary tumors. In this review, we provide an overview of recent progress in the applications of pluripotent stem cells for pituitary research and discuss further perspectives for pituitary studies.

SAT-304 Pituitary Stem Cells May Drive Adenomas Causing Cushing’s Disease

Introduction:Cell rests of self-renewing Sox2+ progenitor cells have been identified in the normal pituitary glands1, however their role in human pituitary tumorigenesis is not understood. Adrenocorticotropic hormone (ACTH) producing microadenomas that cause Cushing’s disease frequently (~70%) lack pathogenic genetic mutations.2 In mice, targeted expression of oncogenic β-catenin in Sox2+ cells generate microadenomas. Interestingly, the Sox2+ cells reside within the adjacent normal gland and drive adenomas in a paracrine fashion.3 We hypothesized that Sox2+ progenitors in human pituitary gland may drive the formation of microadenomas that cause Cushing’s disease (CD). Methods:Four ACTH producing adenomas and two non-functional adenomas (NFPA) with separately annotated adjacent normal tissue (henceforward called ‘microenvironment’) were procured for this study (NCT00060541). We performed RNA deep sequencing (RNAseq) and compared expression of lineage-specific markers and progenitor markers using two-sample T-tests after testing for variance equality and using Welch’s approximation for degrees of freedom. Results:We found expected overexpression of ACTH preprohormone POMC in CD adenomas compared to adjacent microenvironment (?-fold) and NFPA (?-fold). The microenvironment in Cushing’s disease showed increased expression of progenitor markers including SOX2, SOX9, CDH1, GRFA2, and KLF4 compared with microenviroment in NFPA. Likewise, the Cushing’s disease microenvironment showed increased expression ofPOMC (26.98 - fold, P = 0.004) as well as PRLR (FC 17.39, P = 0.006) and GH1 (FC 29.91, P = 0.003) implying that increased Sox2+ progenitors contribute to terminally differentiated corticotrope, lactotroph and somatotroph lineages in-vivo. Conclusions:We report increased expression of several progenitor markers and concomitant elevation in tissues-specific markers in the microenvironment of Cushing’s disease patients. Our results indicate that increased pituitary progenitors in the microenvironment of human corticotropinomas may signal in paracrine fashion and may contribute to the pathogenesis of Cushing’s disease.

Elemental Analysis of Aging Human Pituitary Glands Implicates Mercury as a Contributor to the Somatopause.

Background: Growth hormone levels often decline on aging, and this “somatopause” is associated with muscle and bone loss, visceral adiposity and impaired cardiovascular function. Mercury has been detected in human pituitary glands, so to see if mercury could play a part in the somatopause we measured the proportion of people at different ages who had mercury in their anterior pituitary cells.Materials and methods: Paraffin sections of pituitary glands taken at autopsy from 94 people between the ages of 2 and 99 years were stained for inorganic mercury using autometallography. Pituitary mercury content was classified as none, low (<30% of cells) or high (>30% of cells) in increasing two-decade age groups. Autometallography combined with immunohistochemistry determined which hormone-producing cells contained mercury. Laser ablation-inductively coupled plasma-mass spectrometry was used to confirm the presence of mercury.Results: The proportion of people with low-content pituitary mercury remained between 33 and 42% at all ages. The proportion of people with high-content mercury increased with increasing age, from 0% of people in the 2–20 year group to a peak of 50% of people in the 61–80 years group, followed by a fall to 35% of people in the 81–99 years group. Mercury, when present, was found always in somatotrophs, occasionally in corticotrophs, rarely in thyrotrophs and gonadotrophs, and never in lactotrophs. Laser ablation-inductively coupled plasma-mass spectrometry detected mercury in regions of pituitaries that stained with autometallography.Conclusions: The proportion of people with mercury in their anterior pituitary cells, mostly somatotrophs, increases with aging, suggesting that mercury toxicity could be one factor contributing to the decline in growth hormone levels found in advancing age.

RADIOMORPHOMETRIC ANALYSIS OF HUMAN PITUITARY GLAND DURING THE AGING PROCESS

During life, there are significant changes in the function of the hypothalamic-pituitary axis and its relationship to other endocrine glands in the body, which can affect the external and internal gland morphology. Also, the wide range of pathological changes shows the change in pituitary volume. The knowledge of dimensions and volume of the pituitary gland is very important for the correct diagnosis and prognosis of pituitary diseases. The aim of our study was to perform radiomorphometric analysis of size and volume of human pituitary gland according to age and gender on MRI. Our study represents a retrospective study which included 144 subjects (60 male (41.6 %) and 84 female (58.3 %)) with age range 20-80 years, who underwent magnetic resonance imaging (MRI) of the endocranium. Subjects were divided based on age: first (20-39), second (40-59) and the third group (60-80). Three pituitary diameters were measured: anteroposterior (AP), latero-lateral (LL) and craniocaudal (CC). Pituitary volume (V) was calculated based on the formula: V = AP x LL x CC /2. Our study showed a statistically significant difference for the parameters AP-H (p < 0.037) and AP-A (p < 0.040) in patients of the first and second age group. In addition, the parameter CC showed a statistically significant difference between the first and third age group (p < 0.031). The value of AP-H parameter in females was 10.99 ± 1.16 mm, while in males it was 10.43 ± 1.31 mm, indicating a statistically significant difference for AP-H parameter in both genders (p < 0.008). The AP-N parameter in female subjects was 2.21 ± 0.72 mm, and in male subjects 1.91 ± 0.67 mm, which also indicates that there was a statistically significant difference of AP-N parameter between genders (p < 0.012). The pituitary gland parameter values that were obtained in our study show normal values of pituitary gland dimensions in the Serbian population. During the aging process, the morphology of the pituitary gland changes, AP parameter increases, and parameter CC decreases. In addition, differences between genders were recorded which should be kept in mind during the analyses of pathological conditions or injury of the pituitary gland.

MON-450 Pituitary Antibodies In A Cohort Of Us Service Members With Traumatic Brain Injury

Traumatic brain injury (TBI) causes pituitary dysfunction, mainly featuring GH deficiency. Several mechanisms have been proposed to explain this post-traumatic dyspituitarism, including autoimmunity. We analyzed pituitary antibodies in 100 of US Service Members chosen for a history of TBI and hearing impairment, a concurrence typically seen after blast injuries. Each soldier provided two sera, one before (-1,765 to - 66 days) and one after (10 to 177 days) TBI. All 200 sera were screened by indirect immunofluorescence for binding to a human pituitary gland collected at autopsy. Positive sera were then tested by double indirect immunofluorescence for recognition of cells expressing GH, PRL, ACTH, LH, FSH, TSH, alpha-internexin (INA), vimentin, or glial fibrillary-acidic protein (GFAP). Sera were also tested by ELISA for recognition of native GH or recombinant INA. 63 of 200 sera (31 %) contained pituitary antibodies: of them, 33 (16%) recognized the adenohypophysis, 18 (9 %) the neurohypophysis, and 12 (6%) both lobes. In the adenohypophysis, TSH-secreting cells were the most common target (26 of 45, 58%), followed by INA expressing cells (17 of 45, 37%). In the neurohypophysis, 20 of 30 sera (67 %) recognized INA, 6 (20%) vimentin, and 4 (13%) GFAP-expressing cells. There was no temporal association between pituitary antibodies and TBI: of the sera recognizing TSH-secreting cells, half had antibodies in both the pre- and post-TBI serum (13 of 26, 50%), a quarter (6 of 26, 23%) in the pre-TBI serum, and a quarter (7 of 26, 27%) in the post-TBI serum. Similarly, of the sera recognizing INA-expressing cells, 2 (11.7%) had antibodies in both the pre- and post-TBI serum, 9 (53%) only in the pre-TBI serum, and 6 (35.2%) only in the post-TBI serum. INA antibodies measured by ELISA did not differ between the pre- and post-TBI time points. On the contrary, GH antibodies tended to increase after TBI, although not reaching statistical significance (p= 0.055). The study highlights a preferential recognition of TSH- and INA-expressing cells in TBI patients, which remains to be interpreted in the context of their endocrine dysfunctions. It also suggests that the appearance of GH antibodies after TBI contributes to the GH deficiency typically seen in these patients.

Proteomic Analysis of the Human Anterior Pituitary Gland.

The pituitary function is regulated by a complex system involving the hypothalamus and biological networks within the pituitary. Although the hormones secreted from the pituitary have been well studied, comprehensive analyses of the pituitary proteome are limited. Pituitary proteomics is a field of postgenomic research that is crucial to understand human health and pituitary diseases. In this context, we report here a systematic proteomic profiling of human anterior pituitary gland (adenohypophysis) using high-resolution Fourier transform mass spectrometry. A total of 2164 proteins were identified in this study, of which 105 proteins were identified for the first time compared with high-throughput proteomic-based studies from human pituitary glands. In addition, we identified 480 proteins with secretory potential and 187 N-terminally acetylated proteins. These are the first region-specific data that could serve as a vital resource for further investigations on the physiological role of the human anterior pituitary glands and the proteins secreted by them. We anticipate that the identification of previously unknown proteins in the present study will accelerate biomedical research to decipher their role in functioning of the human anterior pituitary gland and associated human diseases.

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome.

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.