Gonadotropins and Sex Steroid Hormones in Captive-Reared Small Yellow Croaker (Larimichthys polyactis) and Their Role in Female Reproductive Dysfunction.

The seed production of small yellow croaker (SYC) is constrained by reproductive dysfunction in captive-reared females. Reproductive dysfunction is closely linked to endocrine reproductive mechanisms. To better understand the reproductive dysfunction in captive broodstock, functional characterization of gonadotropins (GtHs: follicle stimulating hormone ß subunit, fshß; luteinizing hormone ß subunit, lhß; and glycoprotein α subunit, gpα) and sex steroids (17ß-estradiol, E2; testosterone, T; progesterone; P) was performed using qRT-PCR, ELISA, in vivo, and in-vitro assay. The pituitary GtHs and gonadal steroids levels were significantly higher in ripen fish of both sexes. However, changes in lhß and E2 levels in females were not significant in the developing and ripen stages. Furthermore, GtHs and steroids levels were lower in females compared to males throughout the reproductive cycle. In vivo administration of gonadotropin releasing hormone analogue (GnRHa) significantly increased the expression of GtHs in both dose- and time-related manners. The lower and higher doses of GnRHa led to successful spawning in male and female SYC, respectively. Sex steroids in vitro significantly inhibited the expression of lhß in female SYC. Overall, GtHs were shown to play a vital role in final gonadal maturation, while steroids promoted negative feedback in the regulation of pituitary GtHs. Lower levels of GtHs and steroids might be key components in the reproductive dysfunction of captive-reared female SYC.

Functional Characterization of Three GnRH Isoforms in Small Yellow Croaker Larimichthys polyactis Maintained in Captivity: Special Emphasis on Reproductive Dysfunction.

Fish reproduction is regulated by the brain-pituitary-gonad (BPG) axis where the gonadotropin-releasing hormone (GnRH) plays a central role. Seed production of small yellow croaker (Larimichthys polyactis) is performed using captive-reared broodstock known to undergo reproductive dysfunction, which is connected to endocrinological dysfunction. To determine the endocrinological mechanism of GnRHs in the BPG axis of small yellow croaker, full-length sequences of three GnRH isoforms encoding sbGnRH (GnRH1), cGnRH-II (GnRH2), and sGnRH (GnRH3) were cloned and characterized from brain tissue. qRT-PCR, in vivo, and in vitro experiments were performed for functional characterization. The mRNA expression of GnRH1 in the brain and gonadotropin subunits (GPα, FSHß, and LHß) in the pituitary were significantly higher at the ripen stage during gonadal development and GnRH1 at spawning stage during spawning events. Expression of both GnRH1 and GtH subunits was significantly lower in females than males. GtH subunits were induced at higher concentrations of GnRH1 in vivo and in vitro. Sex-steroids significantly inhibited the GnRH1 expression in vitro in a dose-dependent manner. Taken together, results indicated that GnRH1 plays a key role in gonadal maturation and sex-steroids induced negative feedback in the regulation of GnRH. A lower level of GnRH1 and GtHs might be responsible for reproductive dysfunction in a female small yellow croaker.

Preclinical Efficacy and Safety of an Anti-Human VEGFA and Anti-Human NRP1 Dual-Targeting Bispecific Antibody (IDB0076).

Although bevacizumab (Avastin®) has been approved as an antiangiogenic agent against some cancers, the efficacy is transient and unsatisfactory in other cancers most likely owing to the presence of alternative proangiogenic factors. Therefore, simultaneous blocking of several proangiogenic factors may be a promising strategy for antiangiogenic cancer therapeutics. Accordingly, neuropilin-1 (NRP1) is an attractive target because it serves as a multifunctional receptor for the vascular endothelial growth factor (VEGF) family. Here, we aimed to generate and test an anti-VEGFA and anti-NRP1 dual-targeting bispecific antibody (named as IDB0076) by genetic fusion of an NRP1-targeting peptide to the C-terminus of the bevacizumab heavy chain. Similar to the parental antibody (bevacizumab), IDB0076 suppressed VEGFA-induced migration of human endothelial cells. In contrast, IDB0076 inhibited endothelial-cell migration induced by other angiogenesis growth factors and manifested a more potent antitumor activity than that of bevacizumab in a murine tumor xenograft model. When toxicity was preliminarily evaluated in cynomolgus monkeys, IDB0076 showed no substantial adverse effects, e.g., the absence of noticeable nephrotoxicity, which has previously been documented for the combination therapy of bevacizumab and an anti-NRP1 antibody. Thus, VEGFA-and-NRP1 dual-targeting bispecific antibody IDB0076 may be a potent and safe anticancer agent worthy of further preclinical and clinical studies.

Extremely Well-Differentiated Papillary Thyroid Carcinoma Resembling Adenomatous Hyperplasia Can Metastasize to the Skull: A Case Report.

We describe herein histologic, immunohistochemical, and molecular findings and clinical manifestations of a rare case of an extremely well differentiated papillary thyroid carcinoma (EWD-PTC). Similarly, it is also difficult to diagnose follicular variant papillary thyroid carcinoma (FVPTC), whose diagnosis is still met with controversy. A recently reported entity of well-differentiated tumor of uncertain malignant potential (WDT-UMP) is added to the diagnostic spectrum harboring EWD-PTC and FVPTC. We report this case, because EWD-PTC is different from FVPTC in its papillary architecture, and also from WDT-UMP in its recurrence and metastatic pattern. These morphologically deceptive entities harbored diagnostic difficulties in the past because the diagnosis depended solely on histology. However, they are now diagnosed with more certainty by virtue of immunohistochemical and molecular studies. We experienced a case of EWD-PTC, which had been diagnosed as adenomatous hyperplasia 20 years ago and manifested recurrence with lymph node (LN) metastasis 7 years later. After another 7 years of follow-up, a new thyroid lesion had developed, diagnosed as FVPTC, with LN metastasis of EWD-PTC. One year later, the patient developed metastatic FVPTC in the skull. Immunohistochemically, the EWD-PTC was focally positive for CK19, negative for galectin-3, and focally negative for CD56. Molecular studies revealed BRAF-positivity and K-RAS negativity. The FVPTC in the left thyroid showed both BRAF and K-RAS negativity. In conclusion, EWD-PTC and FVPTC share similar histologic features, but they are different tumors with different molecular biologic and clinical manifestations. A large cohort of EWD-PTC should be included in further study.

Do Helper T Cell Subtypes in Lymphocytic Thyroiditis Play a Role in the Antitumor Effect?

BACKGROUND: Papillary thyroid carcinoma (PTC) is frequently accompanied by lymphocytic thyroiditis (LT). Some reports claim that Hashimoto's thyroiditis (the clinical form of LT) enhances the likelihood of PTC; however, others suggest that LT has antitumor activity. This study was aimed to find out the relationship between the patterns of helper T cell (Th) cytokines in thyroid tissue of PTC with or without LT and the clinicopathological manifestation of PTC. METHODS: Fresh surgical samples of PTC with (13 cases) or without (10 cases) LT were used. The prognostic parameters (tumor size, extra-thyroidal extension of PTC, and lymph node metastasis) were analyzed. The mRNA levels of two subtypes of Th cytokines, Th1 (tumor necrosis factor α [TNF-α], interferon γ [IFN-γ ], and interleukin [IL] 2) and Th2 (IL-4 and IL-10), were analyzed. Because most PTC cases were microcarcinomas and recent cases without clinical follow-up, negative or faint p27 immunoreactivity was used as a surrogate marker for lymph node metastasis. RESULTS: PTC with LT cases showed significantly higher expression of TNF-α (p = .043), IFN-γ (p < .010), IL-4 (p = .015) than those without LT cases. Although the data were not statistically significant, all analyzed cytokines (except for IL-4) were highly expressed in the cases with higher expression of p27 surrogate marker. CONCLUSIONS: These results indicate that mixed Th1 (TNF-α, IFN-γ , and IL-2) and Th2 (IL-10) immunity might play a role in the antitumor effect in terms of lymph node metastasis.

Detection of BRAF V600E mutation with thyroid tissue using pyrosequencing: comparison with PNA-clamping and real-time PCR.

We used pyrosequencing, peptide nucleic acid (PNA)-clamping polymerase chain reaction (PCR), and real-time PCR to detect the BRAF V600E mutation and to investigate the prognostic effect of the BRAF V600E mutation in paraffin block specimens from 100 patients diagnosed with papillary thyroid carcinoma. Positive rates of PNA-clamping PCR, real-time PCR, and pyrosequencing were 66%, 70%, and 68%, respectively. Pyrosequencing and PNA-clamping PCR detected mutant type in a 99:1 (wild-type: mutant) DNA concentration, and PNA-clamping PCR detected mutant type in a 99.5:0.5 DNA concentration. Clamping PCR showed higher κ value than real-time PCR (0.729 vs 0.626). The BRAF V600E mutation was associated with an advanced stage of cancer (P = .045) and was found to be associated with poor prognostic factors. This study suggests that pyrosequencing can be as sensitive as real-time PCR and that PNA-clamping PCR is a sensitive and reliable method to detect the BRAF V600E mutation.

Advanced gastric carcinoma with signet ring cell carcinoma versus non-signet ring cell carcinoma: differentiation with multidetector CT.

OBJECTIVE: The purpose of this study is to assess the capability of multidetector computed tomography (MDCT) to assist in the differentiation of advanced gastric carcinoma with signet ring cell type from that with non-signet ring cell carcinoma (NSRC) with a focus on the thickened stomach wall itself. METHODS: We retrospectively reviewed MDCT results in 80 patients with pathologically proven advanced gastric carcinoma with signet ring cell carcinoma (SRC) (n = 35) and NSRC (n = 45). MDCT images of 80 patients were analyzed retrospectively on gross appearance of thickened gastric wall (polypoid/fungating/ulcerative/diffuse infiltrative), predominantly thickened layer (inner/outer), contrast-enhancement pattern (nonlayered/layered) and degree of enhancement (high/moderate/low). RESULTS: The most common type of gross appearance in both carcinomas was fungating, and the more common contrast-enhancement pattern in both carcinomas was a nonlayered pattern. The predominantly thickened layer was a high attenuation inner layer in both carcinomas. High-degree contrast enhancement was more common in SRC (37.1% of patients) than NSRC (15.6% of patients) with statistically significant difference (P = 0.01). CONCLUSIONS: Multidetector CT cannot distinguish SRC from NSRC based on the thickened stomach wall alone. But, high-degree contrast enhancement was more common in advanced gastric carcinoma with SRC than that with NSRC.

Intranuclear cytoplasmic pseudoinclusions in pituitary adenomas.

Intranuclear pseudoinclusions are well known in papillary carcinomas of the thyroid gland, hepatocellular carcinomas, meningiomas, paragangliomas, pheochromocytomas, and melanomas. Only two papers on the intranuclear inclusions of adenohypophyseal cells in humans have been reported. This study found that intranuclear cytoplasmic pseudoinclusions occur frequently in pituitary adenoma cases (70.3%, 97 of 138 pituitary adenomas) and are uncommon in normal pituitary tissue (11.1%, 1 of 9 normal pituitary tissues). In addition, the frequency of intranuclear cytoplasmic pseudoinclusions between the functional and non-functional pituitary adenomas was found to be similar. Electron microscopy and immunostaining was used to reveal the entity of the intranuclear inclusion. These intranuclear inclusions are due to cytoplasmic invagination because 1) the inclusions are continuous with the cytoplasm, 2) all cytoplasmic organelles, such as the endoplasmic reticulum, the Golgi apparatus, and the secretory granules are found in the inclusions, 3) immunoreactivity of the intranuclear inclusion is the same as that of the cytoplasm. In conclusion, intranuclear cytoplasmic pseudoinclusions in pituitary adenomas occur frequently (70.3%) and are formed by cytoplasmic invagination. This study suggests that pituitary intranuclear inclusions caused by cytoplasmic invagination be called "intranuclear cytoplasmic pseudoinclusions".