Novel imaging techniques in refractory pituitary adenomas.

Accurate localization of the site(s) of active disease is key to informing decision-making in the management of refractory pituitary adenomas when autonomous hormone secretion and/or continued tumor growth challenge conventional therapeutic approaches. In this context, the use of non-standard MR sequences, alternative post-acquisition image processing, or molecular (functional) imaging may provide valuable additional information to inform patient management.

Modern imaging in Cushing's disease.

Management of Cushing's disease is informed by dedicated imaging of the sella and parasellar regions. Although magnetic resonance imaging (MRI) remains the investigation of choice, a significant proportion (30-50%) of corticotroph tumours are so small as to render MRI indeterminate or negative when using standard clinical sequences. In this context, alternative MR protocols [e.g. 3D gradient (recalled) echo, with acquisition of volumetric data] may allow detection of tumors that have not been previously visualized. The use of hybrid molecular imaging (e.g. 11C-methionine positron emission tomography coregistered with volumetric MRI) has also been proposed as an additional modality for localizing microadenomas.

11C-methionine PET aids localization of microprolactinomas in patients with intolerance or resistance to dopamine agonist therapy.

PURPOSE: To assess the potential for 11C-methionine PET (Met-PET) coregistered with volumetric magnetic resonance imaging (Met-PET/MRCR) to inform clinical decision making in patients with poorly visualized or occult microprolactinomas and dopamine agonist intolerance or resistance. PATIENTS AND METHODS: Thirteen patients with pituitary microprolactinomas, and who were intolerant (n = 11) or resistant (n = 2) to dopamine agonist therapy, were referred to our specialist pituitary centre for Met-PET/MRCR between 2016 and 2020. All patients had persistent hyperprolactinemia and were being considered for surgical intervention, but standard clinical MRI had shown either no visible adenoma or equivocal appearances. RESULTS: In all 13 patients Met-PET/MRCR demonstrated a single focus of avid tracer uptake. This was localized either to the right or left side of the sella in 12 subjects. In one patient, who had previously undergone surgery for a left-sided adenoma, recurrent tumor was unexpectedly identified in the left cavernous sinus. Five patients underwent endoscopic transsphenoidal selective adenomectomy, with subsequent complete remission of hyperprolactinaemia and normalization of other pituitary function; three patients are awaiting surgery. In the patient with inoperable cavernous sinus disease PET-guided stereotactic radiosurgery (SRS) was performed with subsequent near-normalization of serum prolactin. Two patients elected for a further trial of medical therapy, while two declined surgery or radiotherapy and chose to remain off medical treatment. CONCLUSIONS: In patients with dopamine agonist intolerance or resistance, and indeterminate pituitary MRI, molecular (functional) imaging with Met-PET/MRCR can allow precise localization of a microprolactinoma to facilitate selective surgical adenomectomy or SRS.

A Novel Thyrotropin-Releasing Hormone Receptor Missense Mutation (P81R) in Central Congenital Hypothyroidism.

CONTEXT: Isolated central congenital hypothyroidism (CCH) is rare and evades diagnosis on TSH-based congenital hypothyroidism (CH) screening programs in the United Kingdom. Accordingly, genetic ascertainment facilitates diagnosis and treatment of familial cases. Recognized causes include TSH ß subunit (TSHB) and Ig superfamily member 1 (IGSF1) mutations, with only two previous reports of biallelic, highly disruptive mutations in the TRH receptor (TRHR) gene. CASE DESCRIPTION: A female infant presenting with prolonged neonatal jaundice was found to have isolated CCH, with TSH of 2.2 mU/L (Reference range, 0.4-3.5) and free T4 of 7.9 pmol/L (0.61 ng/dL) (Reference range, 10.7-21.8 pmol/L). Because TSHB or IGSF1 mutations are usually associated with profound or X-linked CCH, TRHR was sequenced, and a homozygous mutation (p.P81R) was identified, substituting arginine for a highly conserved proline residue in transmembrane helix 2. Functional studies demonstrated normal cell membrane expression and localization of the mutant TRHR; however, its ability to bind radio-labelled TRH and signal via Gqα was markedly impaired, likely due to structural distortion of transmembrane helix 2. CONCLUSIONS: Two previously reported biallelic, highly disruptive (nonsense; R17*, in-frame deletion and single amino acid substitution; p.[S115-T117del; A118T]) TRHR mutations have been associated with CCH; however, we describe the first deleterious, missense TRHR defect associated with this phenotype. Importantly, the location of the mutated amino acid (proline 81) highlights the functional importance of the second transmembrane helix in mediating hormone binding and receptor activation. Future identification of other naturally occurring TRHR mutations will likely offer important insights into the molecular basis of ligand binding and activation of TRHR, which are still poorly understood.

Cell division, synthetic capacity and apoptosis in periodontal lesions analysed by in situ hybridisation and immunohistochemistry.

In this study, we investigated the synthetic and proliferative activity of infiltrating mononuclear cells in sections of granulation tissue from periodontitis lesions in both adult periodontitis (AP) and early onset periodontitis (EOP) patients. We also investigated the role of apoptosis in the remodelling of the inflamed tissue. We utilised a Ki-67 antigen specific antibody and a histone messenger RNA (mRNA) probe to detect cells undergoing cell division in the sections. Oligonucleotide probes for 28S ribosomal RNA and for the detection of poly A mRNA were utilised to detect cells with synthetic capacity. Apoptosis was determined using terminal transferase labelling of fragmented DNA with Biotin labelled dUTP. Biopsies of granulation tissue were obtained from 9 AP patients, from 10 EOP patients and for comparative purposes, biopsies of gingival tissue from 4 patients with AP. There were no differences regarding the relative proportions of cells with synthetic capacity or in the numbers of dividing cells in the periodontitis tissue sections. However, we observed an increase in the number of dividing cells in the AP granulation tissues compared to the AP gingival sections and that these cells were predominantly fibroblast like in appearance. Apoptotic cells consisted mainly of connective tissue cells; mainly fibroblasts with few if any leukocytes being apoptotic other than polymorphonuclear leukocytes. Only a few cyto-phagocytic macrophages were ever observed in the gingival and granulation tissues. We conclude that the turnover of infiltrating leukocytes in inflamed periodontal tissue is low, that they probably arrive at this site by recruitment from distant lymph nodes, and that neither cell division nor programmed cell death significantly alter the numbers of inflammatory cells. On the other hand, fibroblast apoptosis and cell division occur within the periodontium as these are typical processes in the normal turnover and remodelling of these tissues.

Humoral immune responses in periodontal disease may have mucosal and systemic immune features.

The humoral immune response, especially IgG and IgA, is considered to be protective in the pathogenesis of periodontal disease, but the precise mechanisms are still unknown. Immunoglobulins arriving at the periodontal lesion are from both systemic and local tissue sources. In order to understand better the local immunoglobulin production, we examined biopsy tissue from periodontitis lesions for the expression of IgM, IgG, IgA, IgE and in addition the IgG and IgA subclasses and J-chain by in situ hybridization. Tissues examined were superficial inflamed gingiva and the deeper granulation tissue from periodontal sites. These data confirm that IgM, and IgG and IgA subclass proteins and J-chain can be locally produced in the periodontitis tissues. IgG1 mRNA-expressing cells were predominant in the granulation tissues and in the gingiva, constituting approx. 65% of the total IgG-expressing plasma cells. There was a significantly increased proportion of IgA-expressing plasma cells in the gingiva compared with the granulation tissue (P < 0.01). Most of the IgA-expressing plasma cells were IgA1, but a greater proportion expressed IgA2 mRNA and J-chain mRNA in the gingival tissues (30.5% and 7.5%, respectively) than in the periodontal granulation tissues (19% and 0-4%, respectively). The J-chain or dimeric IgA2-expressing plasma cells were located adjacent to the epithelial cells, suggesting that this tissue demonstrates features consistent with a mucosal immune response. Furthermore, we were able to detect the secretory component in gingival and junctional epithelial cells, demonstrating that the periodontal epithelium shares features with mucosal epithelium. In contrast, deeper tissues had more plasma cells that expressed IgM, and less expressing IgA, a response which appears more akin to the systemic immune response. In conclusion, this study suggests that immune mechanisms involved in the pathogenesis of periodontitis may involve features of both the mucosal and systemic immune systems, dependent on tissue location.

Relative proportions of mononuclear cell types in periodontal lesions analyzed by immunohistochemistry.

In this study, we investigated the relative proportions of infiltrating mononuclear inflammatory cells in sections of granulation tissue from periodontitis lesions in both adult periodontitis (AP) and early onset periodontitis (EOP) patients. We utilised a set of cluster of differentiation (CD) antigen-specific monoclonal antibodies to detect different cell types within the tissues. These included anti-CD 20 (B cells), anti-CD 3 (pan T cells) and anti-CD 45RO (memory T cells), anti-CD 4 (helper T cells) anti-CD 8 (suppressor T cells) and anti-CD 68 (monocyte/macrophage). Biopsies of granulation tissue were obtained from 9 patients with adult periodontitis (AP), from 10 patients with early onset periodontitis (EOP) and for comparative purposes, biopsies of gingival tissue from 4 patients with AP. A significantly greater number of T cells (p < 0.05) were observed in EOP and gingival sections than in AP sections. In addition, a greater number of B cells were observed in the granulation tissues than in the gingiva (p < 0.05). The relative numbers of B cells (CD 20). T cells (CD 3) and macrophages (CD 68) were expressed as a percentage of their combined total for each of the patient groups and indicated that the proportion of B lymphocytes was greater in AP sections than in EOP or gingival sections (p < 0.02). The proportion of T cells was lower in the AP periodontitis sections than in the EOP periodontitis sections (p < 0.05). There were no significant differences in the proportion of macrophages between the 3 categories of tissue specimens. The relative ratios of B cells (CD 20) to T cells (CD 3) and B cells (CD 20) to memory T cells (CD 45RO) and macrophages (CD 68) to T cells (CD 3) and memory T cells (CD 45RO) were analyzed and indicated that there was a significant increase in the B to T cell ratio in AP sections compared to EOP and gingival sections (p < 0.02). There was also a significant increase in the macrophage to T cell ratio in AP sections as indicated by CD 68 to CD 3 ratios (p < 0.05). There were no differences regarding the relative proportions of memory T cells or in the ratios of CD 4+ to CD 8+ T cells in the different disease categories. In conclusion, these differences in the relative proportions of B cells, T cells and macrophages may reflect a difference in the immunopathology of AP and EOP.