Aldosterone-Producing Adenomas of Increased Size Are Associated With Higher Steroidogenic Activity.

CONTEXT: There are inconsistent results and insufficient evidence as to whether an association exists between the size and aldosterone-producing ability of aldosterone-producing adenomas. OBJECTIVE: We further investigated this possible association retrospectively. METHODS: A total of 142 cases of primary aldosteronism diagnosed as unilateral by adrenal venous sampling at 2 referral centers between 2009 and 2019 were included. We classified these individuals into small and large tumor groups using a diameter of 14 mm as a cutoff. This size was the median diameter of the tumor on the affected side of the adrenal gland. We compared plasma aldosterone concentration (PAC), plasma renin activity (PRA), PAC to PRA ratio, PAC from a saline infusion test (SIT), urinary aldosterone secretion (uAld), and serum potassium as indices of aldosterone-producing ability between the 2 groups. In some cases, we conducted histopathological evaluations and detection of the KCNJ5 mutation. RESULTS: PAC, PAC to PRA ratio, PAC from SIT, and uAld were higher and serum potassium was lower in the large tumor group. PAC, PAC from SIT, uAld, and serum potassium significantly correlated with tumor diameter. PRA was not associated with tumor diameter. Clear cell-dominant cases were more common in the large tumor group, while cases showing a strong expression of CYP11B2 were not significantly different between the groups. KCNJ5 mutations tended to be more common in the large tumor group. CONCLUSION: The higher aldosterone-producing ability in larger adenomas can be used to infer the responsible lesion and disease type.

Identification of interleukin-16 production on tumor aggravation in hepatocellular carcinoma by a proteomics approach.

BACKGROUND: Cytokines play an important role in the immune response, angiogenesis, cell growth, and differentiation in hepatocellular carcinoma (HCC). OBJECTIVE: We performed a comprehensive study to identify tumor-related cytokines and pathways involved in HCC pathogenesis. METHODS: Cytokine production was evaluated in human HCC tissues and adjacent non-tumor tissues using an antibody-based protein array technique. We compared cytokine expression in HCC tissues with that of hepatic hemangioma (HH), liver metastasis of colorectal cancer, and noncancerous liver tissues from transplantation donors. The protein levels and localization of the candidate cytokines were analyzed by western blotting and immunohistochemistry. RESULTS: Increased expression of interleukin (IL)-1 receptor antagonist, macrophage migration inhibitory factor, and IL-16 was observed in HCC and paired adjacent non-tumor tissues compared with noncancerous livers. In addition, there were increased IL-16 levels in HCC tissues compared with HH. IL-16 treatment significantly increased cell proliferation in vitro. The expression of extracellular signal-regulated kinase (ERK)1/2 and cyclin D1 was markedly increased in cells from two HCC cell lines, Huh7 and HepG2, in a dose- and time-dependent manner. Phosphorylated to total ERK1/2 ratio was increased in Huh7 cells following IL-16 50 ng/ml, but not HepG2 cells. ERK phosphorylation have occurred earlier than protein accumulation at 48 h. Pretreatment with the ERK inhibitor, FR18024, or an anti-IL-16 antibody reduced the increase in IL-16 production in HCC cells. CONCLUSIONS: These results suggest that cell proliferation induced by IL-16 is mediated through the ERK pathway, thus, we identified a new factor associated with HCC tumor growth.

Immune checkpoint inhibitor combination therapies very frequently induce secondary adrenal insufficiency.

Immune checkpoint inhibitors (ICIs) are potent therapeutic options for many types of advanced cancer. The expansion of ICIs use however has led to an increase in immune-related adverse events (irAEs). Secondary adrenal insufficiency (AI) can be life-threatening especially in patients with delayed diagnosis. We retrospectively investigated secondary AI in ICI-treated patients. A total of 373 cancer patients treated with ICIs were included and evaluated. An adrenocorticotropic hormone (ACTH) deficiency was described in 13 patients. Among 24 patients with a combination of nivolumab and ipilimumab therapy, 7 patients (29%) developed secondary AI in a median time of 8 weeks during the combination therapy and 2 of 15 patients (13%) developed isolated ACTH deficiency during maintenance nivolumab monotherapy following the combination therapy. More than half of the patients (4/7) with a combination therapy-induced multiple anterior hormone deficiencies was diagnosed as secondary AI based on regular ACTH and cortisol tests with slight subjective symptoms. Secondary AI can arise frequently and rapidly in cancer patients receiving a combination ICI therapy, and thus we speculate active surveillance of AI using regular ACTH and cortisol tests during the combination therapy might be useful for avoiding life-threatening conditions due to secondary AI.

Bexarotene-induced hypothyroidism: Characteristics and therapeutic strategies.

OBJECTIVE: Central hypothyroidism (CH) is a well-known adverse effect of bexarotene treatment for cutaneous T-cell lymphoma (CTCL). While concomitant levothyroxine therapy is recommended in these cases, associations between ethnic variation or susceptibility and bexarotene-induced CH have not yet been reported. This study aimed to characterize the kinetics and dose dependency of bexarotene-induced CH in Japanese patients. DESIGN AND PATIENTS: Sixty-six Japanese patients with CTCL were retrospectively investigated by evaluating thyroid function during the early phase of bexarotene therapy. RESULTS: At one week after bexarotene initiation, TSH and FT4 values significantly declined. However, this effect was not bexarotene dose-dependent at least at the dose of 96-320 mg/m2 . Approximately 1 month later, 61 patients exhibited hypothyroidism at a relatively low dose of bexarotene (average 251 mg/m2 /day). Forty-five study cases showed this effect at 1 week. Simple regression analyses indicated that higher pretreatment TSH values (at a cut-off value of 1.30:73% sensitivity, 57% specificity) or lower normal (within the lower half of the reference range) pretreatment FT4 values (84% sensitivity, 57% specificity) were predictive of hypothyroidism at 1 week. The remaining 21 cases showed euthyroidism at 1 week, at which TSH values may roughly predict their thyroid function at 1 month (at a cut-off value of 0.05:100% sensitivity, 80% specificity). CONCLUSIONS: Preventive treatment with levothyroxine is recommended for Japanese CTCL patients prior to bexarotene therapy. Minimally, it should be considered for patients with a pretreatment TSH above 1.30, a lower normal pretreatment FT4, or a TSH below 0.05 at 1 week.

Molecular analysis and literature-based hypothesis of an immunonegative prostate small cell carcinoma causing ectopic ACTH syndrome.

Ectopic ACTH syndrome (EAS) due to a prostate small cell carcinoma (SCC) is very rare with only 26 cases reported to date and has a poor prognosis. We here describe another case of this disorder that was clinically typical based on prior reports as it showed hypercortisolemia and severe hypokalemia with multiple metastasis. However, our current case of prostate SCC causing EAS is the first to display negative immunostaining for ACTH despite detectable POMC mRNA expression in the primary lesion. ACTH immunonegativity is thought to be associated with a more aggressive disease course and a poorer prognosis although there are few studies of the underlying mechanisms. We explored two possibilities for this finding in our current patient: aberrant POMC processing prevented immunodetection with an anti-ACTH antibody; and the ACTH content per cell was below the threshold for immunodetection due to its rapid secretion or low synthesis. The aberrant processing theory was thought to be less likely because of immunonegative findings even using anti-POMC/ACTH antibodies. As the plasma ACTH levels in our patient were comparable with those reported for previous immunopositive prostate EAS cases, we speculated that the depletion of ACTH may be caused not only by rapid secretion but also by low production levels as a sign of de-differentiation. De-differentiation may therefore explain the mechanism underlying the negative correlation between immunoreactivity for ACTH in EAS and disease aggressiveness. We believe that our present findings will be of use in future prospective studies aimed at confirming the mechanism of immunonegativity.

Vasoactive intestinal peptide increases apoptosis of hepatocellular carcinoma by inhibiting the cAMP/Bcl-xL pathway.

Vasoactive intestinal peptide (VIP) is a modulator of inflammatory responses. VIP receptors are expressed in several tumor types, such as colorectal carcinoma. The study described herein was conducted to confirm the presence of VIP and its receptors (VPAC1 and VPAC2) in surgically resected hepatocellular carcinoma (HCC) tissues and in the HCC cell line Huh7. The mechanism responsible for apoptosis of HCC cells was then examined because VIP treatment (10-10  M) significantly suppressed proliferation of Huh7 cells. In examining apoptosis-related proteins, we found caspase-3 to be significantly increased and Bcl-xL and cyclic AMP (cAMP) response element-binding protein (CREB) to be significantly decreased in Huh7 cells cultured with VIP. Furthermore, the CREB level and phosphorylation were reduced. These effects were reversed by the addition of VIP receptor antagonist or cAMP antagonist Rp-cAMPS. Pretreatment with cAMP analogue blocked the increased apoptosis, suggesting that VIP induces apoptosis via a PKA-independent signaling mechanism. Our data indicate that VIP prevents the progression of HCC by apoptosis through the cAMP/Bcl-xL pathway.

Isolation of Adipose-Derived Stem/Stromal Cells from Cryopreserved Fat Tissue and Transplantation into Rats with Spinal Cord Injury.

Adipose tissue contains multipotent cells known as adipose-derived stem/stromal cells (ASCs), which have therapeutic potential for various diseases. Although the demand for adipose tissue for research use remains high, no adipose tissue bank exists. In this study, we attempted to isolate ASCs from cryopreserved adipose tissue with the aim of developing a banking system. ASCs were isolated from fresh and cryopreserved adipose tissue of rats and compared for proliferation (doubling time), differentiation capability (adipocytes), and cytokine (hepatocyte growth factor and vascular endothelial growth factor) secretion. Finally, ASCs (2.5 × 106) were intravenously infused into rats with spinal cord injury, after which hindlimb motor function was evaluated. Isolation and culture of ASCs from cryopreserved adipose tissue were possible, and their characteristics were not significantly different from those of fresh tissue. Transplantation of ASCs derived from cryopreserved tissue significantly promoted restoration of hindlimb movement function in injured model rats. These results indicate that cryopreservation of adipose tissue may be an option for clinical application.

Intravenous infusion of adipose-derived stem/stromal cells improves functional recovery of rats with spinal cord injury.

BACKGROUND AIMS: Adipose tissue has therapeutic potential for spinal cord injury (SCI) because it contains multipotent cells known as adipose-derived stem/stromal cells (ASCs). In this study, we attempted intravenous ASC transplantation in rats with SCI to examine the effect on functional recovery. METHODS: ASCs (2.5 × 106) were intravenously infused into SCI rats, after which hindlimb motor function was evaluated. Distribution of transplanted ASCs was investigated and growth factor/cytokine levels were determined. RESULTS: Intravenous transplantation of ASCs promoted the functional recovery in SCI rats and reduced the area of spinal cord cavitation. A distribution study revealed that ASCs gradually accumulated at the site of injury, but long-term survival of these cells was not achieved. Levels of growth factors increased only slightly in the spinal cord after ASC transplantation. Unexpectedly, cytokine-induced neutrophil chemoattractant (CINC)-1 showed a transient but substantial increase in the spinal cord tissue and blood of the ASC group. CINC-1 was secreted by ASCs in vitro, and the sponge implantation assay showed that CINC-1 and ASCs induced angiogenesis. CINC-1 promoted functional recovery in SCI rats, which was similar to the ASCs. Expression of glial cell line-derived neurotrophic factor was greater in the ASC group than in the CINC-1 group, although both promoted extracellular signal-regulated kinase (ERK)1/2 phosphorylation; Akt phosphorylation was enhanced in the spinal cord after ASC transplantation. CONCLUSIONS: Our findings indicated that intravenously transplanted ASCs gradually accumulated in the injured spinal cord, where cytokines such as CINC-1 activated ERK1/2 and Akt, leading to functional recovery.

Erdheim-Chester disease and pituitary involvement: a unique case and the literature.

An early thirties man diagnosed with Erdheim-Chester disease (ECD) was simultaneously disclosed to have hypogonadotropic hypogonadism, central adrenal insufficiency, and GH deficiency in addition to central diabetes insipidus (CDI). Pituitary magnetic resonance imaging (MRI) showed swelling in the stalk, enlargement of the anterior lobe with delayed enhancement, and loss of high intensity of the posterior lobe on T1-weighted images, suggesting of pituitary involvement of ECD. Three months after starting treatment with interferon α and zoledronic acid, polyuria and polydipsia were ameliorated without DDAVP, accompanied with improvement of MRI. Simultaneously technetium-99m bone scintigraphy showed improvement, accompanied with a relief of bone pain and high fever. In contrast, he developed secondary hypothyroidism with slight enlargement of anterior pituitary gland without relapse of CDI, suggesting of different responses to treatment with interferon α between anterior pituitary lobe and posterior one. So far he continues to be replaced with deficient hormone replacement therapy. As for bone pain, it remains to be controlled with the decreased levels of bone resorption marker with decreased abnormal uptake in bone scintigraphy although zoledronic acid was discontinued for osteonecrosis of the jaw. For four years, he has not showed new involvement at other organs besides bones and the pituitary. While CDI is known to be very common in ECD, improvement of CDI has been reported in a few cases. Other endocrine manifestations, especially with detailed endocrine status, have been also reported in limited cases. Thus we report this case and review the literature.

Tyrosine kinase inhibitor-induced thyroid disorders: a review and hypothesis.

BACKGROUND: Thyroid dysfunction is a well-known adverse effect of sunitinib, a drug that targets multiple receptor tyrosine kinases, including vascular endothelial growth factor receptor (VEGFR). As several kinds of tyrosine kinase inhibitors (TKIs) are now available, this has been postulated to be a side effect of the TKIs that target the VEGFR (VEGF-TKIs). However, sunitinib, one of the first-generation TKIs, likely causes thyroid dysfunction more frequently than other TKI classes, leading not only to hypothyroidism, but also to thyrotoxicosis. SUMMARY: Based on the reports published to date, including our own studies, we have hypothesized that sunitinib may exert these effects, because it targets a broad spectrum of tyrosine kinases. This not only includes VEGFR2, but also VEGFR1 and the platelet-derived growth factor receptor (PDGFR). This, in turn, may suggest that not only VEGFR2 but also the PDGFR and/or the VEGFR1 play an important role during angiogenesis in the thyroid. CONCLUSIONS: Our current hypothesis may explain the mechanisms that underlie TKI-induced thyroid disorders. By learning how various kinds of TKIs affect thyroid function, we may elucidate how the angiogenesis in thyroid is regulated both physiologically and pathologically.

Regulation of RhoA signaling by the cAMP-dependent phosphorylation of RhoGDIα.

RhoA plays a pivotal role in regulating cell shape and movement. Protein kinase A (PKA) inhibits RhoA signaling and thereby induces a characteristic morphological change, cell rounding. This has been considered to result from cAMP-induced phosphorylation of RhoA at Ser-188, which induces a stable RhoA-GTP-RhoGDIα complex and sequesters RhoA to the cytosol. However, few groups have shown RhoA phosphorylation in intact cells. Here we show that phosphorylation of RhoGDIα but not RhoA plays an essential role in the PKA-induced inhibition of RhoA signaling and in the morphological changes using cardiac fibroblasts. The knockdown of RhoGDIα by siRNA blocks cAMP-induced cell rounding, which is recovered by RhoGDIα-WT expression but not when a RhoGDIα-S174A mutant is expressed. PKA phosphorylates RhoGDIα at Ser-174 and the phosphorylation of RhoGDIα is likely to induce the formation of a active RhoA-RhoGDIα complex. Our present results thus reveal a principal molecular mechanism underlying G(s)/cAMP-induced cross-talk with G(q)/G(13)/RhoA signaling.

Long-term clinical course of IgG4-related systemic disease accompanied by hypophysitis.

A 70-year old man with a 14 year history of Sjögren syndrome, interstitial pneumonia, and autoimmune hepatitis (AIH) was admitted to our hospital due to hyponatremia with a one month history of fatigue, thirst, and nausea. Laboratory tests on admission revealed that this patient had a central adrenal insufficiency. Pituitary magnetic resonance imaging (MRI) further showed swelling of the stalk and posterior lobe of his pituitary, suggesting infundibulo-hypophysitis. Based on his past history of autoimmune disease, his serum IgG4 levels were measured and found to be remarkably high (924 mg/ dL). Previous biopsy specimens from his liver, lung, and parotid gland were immunostained for IgG4, which revealed a marked infiltration of IgG4-positive plasma cells. As a result of our tests, we made a diagnosis of IgG4-related systemic disease. Interestingly, a subsequent MRI scan at three weeks after the patient commenced glucocorticoid replacement therapy for adrenal insufficiency showed that the swelling of his pituitary stalk was reduced. This finding suggested that IgG4-related hypophysitis may improve either as a result of a supplemental dose of glucocorticoid or possibly spontaneously. Although six cases of IgG4-related hypophysitis have been reported in the scientific literature published in English, our current case is the first in which IgG4-related hypophysitis likely occurred as a result of a long-term history of IgG4-related systemic disease. We report this case herein and review the relevant literature.

Sunitinib induces hypothyroidism with a markedly reduced vascularity.

BACKGROUND: Sunitinib is a small molecule that inhibits receptor tyrosine kinases, including the vascular endothelial growth factor receptors, and exhibits antiangiogenic and antitumor activity. This molecule has also been reported to cause hypothyroidism at a high frequency, but the mechanism of this is unknown. SUMMARY: A 60-year-old woman was administered sunitinib for the treatment of metastatic renal cell carcinoma. One week later, she displayed overt hypothyroidism with an atrophic thyroid and a marked reduction in vascularity as determined by ultrasonography, despite high levels of thyrotropin. In contrast, during the off-periods in the sunitinib treatment cycles, the volume of her thyroid recovered with an increase in vascularity despite a low level of thyrotropin. These results suggest that thyroid function and volume may depend on the vascularity, which is negatively regulated by sunitinib. CONCLUSION: Our case study provides compelling evidence that sunitinib induces hypothyroidism by reducing blood flow via capillary regression and constriction.

Human G(salpha) mutant causes pseudohypoparathyroidism type Ia/neonatal diarrhea, a potential cell-specific role of the palmitoylation cycle.

Pseudohypoparathyroidism type Ia (PHP-Ia) results from the loss of one allele of G(salpha), causing resistance to parathyroid hormone and other hormones that transduce signals via G(s). Most G(salpha)mutations cause the complete loss of protein expression, but some cause loss of function only, and these have provided valuable insights into the normal function of G proteins. Here we have analyzed a mutant G(salpha) (alphas-AVDT) harboring AVDT amino acid repeats within its GDP/GTP binding site, which was identified in unique patients with PHP-Ia accompanied by neonatal diarrhea. Biochemical and intact cell analyses showed that alphas-AVDT is unstable but constitutively active as a result of rapid GDP release and reduced GTP hydrolysis. This instability underlies the PHP-Ia phenotype. alphas-AVDT is predominantly localized in the cytosol, but in rat and mouse small intestine epithelial cells (IEC-6 and DIF-12 cells) alphas-AVDT was found to be localized predominantly in the membrane where adenylyl cyclase is present and constitutive increases in cAMP accumulation occur in parallel. The likely cause of this membrane localization is the inhibition of an activation-dependent decrease in alphas palmitoylation. Upon the overexpression of acyl-protein thioesterase 1, however, alphas-AVDT translocates from the membrane to the cytosol, and the constitutive accumulation of cAMP becomes attenuated. These results suggest that PHP-Ia results from the instability of alphas-AVDT and that the accompanying neonatal diarrhea may result from its enhanced constitutive activity in the intestine. Hence, palmitoylation may control the activity and localization of G(salpha) in a cell-specific manner.

An instructive case suggesting cyclical primary hyperparathyroidism.

We report an instructive case of primary hyperparathyroidism in which cyclical secretion of PTH may have caused repeated hypercalcemic crises followed by temporary remission with a spontaneous drop in PTH. A 64-year-old man was admitted to our hospital twice with severe hypercalcemic crisis (corrected calcium (cCa) 15.0 mg/dl and 16.7 mg/dl) accompanied by an increase in intact PTH (220 pg/ml and 470 pg/ml). During both events, the serum PTH values spontaneously dropped followed by remission of the hypercalcemia. The tumor, detected at the left-upper side, showed neither vascularity on ultrasound examination nor washout delay on MIBI scintigraphy, suggesting that two parathyroid adenoma infarctions had occurred. Cervical exploration was undertaken. The histopathological examinations confirmed that the tumor was parathyroid adenoma. Contrary to our expectation, however, it did not reveal necrotic tissue that would indicate recent infarction. The findings in this case may be explained by cyclical secretion of PTH from a parathyroid adenoma. Although cyclical Cushing's syndrome is well known, to our knowledge this is the first documented case suggesting cyclical primary hyperparathyroidism.

Roles of CTPL/Sfxn3 and Sfxn family members in pancreatic islet.

Pancreatic AR42J cells have the feature of pluripotency of the precursor cells of the gut endoderm. Betacellulin (BTC) and activin A (Act) convert them into insulin-secreting cells. Using mRNA differential display techniques, we have identified a novel mitochondrial transporter, which is highly expressed during the course of differentiation, and have designated it citrate transporter protein-like protein (CTPL). Recently sideroflexin 1 (Sfxn1) was shown to be a susceptible gene of flexed-tail (f/f) mice, and CTPL has turned out to be a rat orthologous protein of Sfxn3, a member of sideroflexin family. CTPL/Sfxn3 was targeted to mitochondrial membrane like Sfxn1. The expression levels of CTPL/Sfxn3, Sfxn2, and Sfxn5 were upregulated in the early phase of differentiation into insulin-secreting cells but the expression levels of Sfxn1 and Sfxn3 did not change. All Sfxn family members were expressed in rat pancreatic islet. The expression levels of CTPL/Sfxn3, Sfxn2, and Sfxn5 were also upregulated in islets of streptozotocin-induced diabetic rats compared to normal rats. The downregulation of CTPL/Sfxn3 in a rat insulinoma cell line, INS-1, with the antisense oligonucleotide did not affect the insulin secretion. Taken together, CTPL/Sfxn3 and some other family members might be important in the differentiation of pancreatic beta-cells as a channel or a carrier molecule and be related to the regeneration of pancreatic endocrine cells.

Mechanical stress activates angiotensin II type 1 receptor without the involvement of angiotensin II.

The angiotensin II type 1 (AT1) receptor has a crucial role in load-induced cardiac hypertrophy. Here we show that the AT1 receptor can be activated by mechanical stress through an angiotensin-II-independent mechanism. Without the involvement of angiotensin II, mechanical stress not only activates extracellular-signal-regulated kinases and increases phosphoinositide production in vitro, but also induces cardiac hypertrophy in vivo. Mechanical stretch induces association of the AT1 receptor with Janus kinase 2, and translocation of G proteins into the cytosol. All of these events are inhibited by the AT1 receptor blocker candesartan. Thus, mechanical stress activates AT1 receptor independently of angiotensin II, and this activation can be inhibited by an inverse agonist of the AT1 receptor.