Immunohistochemical evaluation of tumor budding for stratifying T1 colorectal cancer: optimal cut-off value and a novel computer-assisted semiautomatic method.

High-grade tumor budding is an adverse prognostic factor for submucosal invasive (T1) colorectal cancer used to predict the risk for lymph node metastasis in endoscopically resected specimens. Cytokeratin immunohistochemistry is a potential option for evaluating tumor budding. The optimal cut-off value between low- and high-grade budding has not yet been determined, however, and the high inter-observer variability in selecting budding foci remains problematic. We explored the optimal cut-off value for predicting lymph node metastasis using cytokeratin immunohistochemistry, and developed a novel computer-assisted semiautomatic quantification method to reduce inter-observer variability. A retrospective single-institution study of 463 T1 colorectal cancer cases was conducted. Cases were split into derivation and validation datasets. Tumor budding foci were counted manually and semiautomatically using Image J software on cytokeratin immunohistochemistry-stained specimens. We determined the cut-off values and compared inter-observer variability among pathologists between the two methods. Univariate and multivariate analyses of the derivation dataset were performed to select the risk factors for lymph node metastasis. Predictive simulation for the validation dataset was conducted. The optimal cut-off values for the manual and semiautomatic methods were ≥10 and ≥12, respectively. For both methods, multivariate analyses revealed that venous invasion, lymphatic invasion, and high-grade tumor budding were independent risk factors for lymph node metastasis. The semiautomatic method provided significantly better inter-observer agreement. The predictive and observed lymph node metastasis frequencies were highly correlated in the validation dataset.

Virilism and Ectopic Expression of HSD17B5 in Mature Cystic Teratoma.

Mature cystic teratoma (MCT) is rarely involved in the overproduction of steroid hormones in contrast to sex cord stromal tumors. A 31-year-old woman visited our hospital with hirsutism, hoarseness, and hair loss from the scalp. Serum testosterone and free-testosterone levels were 7.3 ng/ml and 2.3 pg/ml, respectively, which were markedly in excess of the age adjusted female standard levels. Basal blood levels of steroid hormones and serum levels of 17-hydroxyprogesterone at 1 h after intravenous injection of adrenocorticotropic hormone demonstrated that 21-hydroxylase deficiency was not the underlying cause of her virilization. A subsequent chromosomal test with G-banding revealed a karyotype of 46XX. Magnetic resonance imaging revealed a mass in the left ovary, which was subsequently diagnosed as MCT. Detailed pathological analysis of the tumor indicated that it was comprised of skin components, sweat glands, with hair and fat texture, glandular epithelium and fibrous connective tissue, consistent with the characteristic composition of MCT. Immunohistochemical analysis demonstrated marked immunoreactivity of 17beta-hydroxysteroid dehydrogenase (HSD17B5), an enzyme that can convert androstenedione to testosterone. Following surgical removal of the tumor, testosterone and free testosterone levels were markedly decreased (0.3 ng/ml and 0.4 pg/ml, respectively) and other symptoms abated. In conclusion, this is the first report of an ovarian MCT associated with clinical virilization caused by the ectopic production of testosterone possibly because of an overexpression of intratumoral HSD17B5.

The impact of inflow treatment for claudicants with both aortoiliac and femoropopliteal occlusive disease.

PURPOSE: This study evaluated the early and long-term results of the sole use of endovascular treatment in the treatment of inflow lesions in claudicants with both aortoiliac and femoropopliteal (FP) lesions. METHODS: A retrospective study that included 100 limbs in 73 patients was performed. The patency rates of aortoiliac artery stents, the continued clinical improvement rates, the risk factors for persistent disabling claudication after inflow revascularization, and the rates of freedom from additional FP procedures were examined. RESULT: After inflow revascularization, almost complete relief from intermittent claudication was seen in 79 % of the limbs, while 21 % of the limbs continued to suffer from disabling claudication. A multivariate analysis showed that a run-off score of ≥7 was an independent predictor for persistent disabling claudication after aortoiliac revascularization [hazard ratio (HR) 5.11, 95 % confidence interval (CI) 1.34-19.45; P = 0.02]. The primary patency rates at 1, 3, 5, and 6 years were 96, 96, 96 and 89 %, respectively. The secondary patency rate at 6 years was 100 %. The continued clinical improvement rates at 1, 3, 5, and 6 years were 81, 78, 78 and 72 %, respectively. The rates of freedom from additional FP procedures at 1, 3, 5, and 6 years were 97, 90, 90, and 90 %, respectively. CONCLUSIONS: Aortoiliac endovascular revascularization is effective treating claudicants with both aortoiliac and femoropopliteal lesions. Furthermore, a run-off score of ≥7 appears to be a potential predictor for persistent disabling claudication.

A case of Cushing's syndrome due to bilateral cortisol-secreting adenomas with unilateral DHEAS oversecretion.

A 39-year-old Japanese woman presented with typical clinical symptoms of Cushing's syndrome, including amenorrhea and hirsutism, for 2 years. The results of her initial endocrine evaluation were consistent with ACTH-independent Cushing's syndrome due to bilateral adrenal masses (diameters of 3.1 cm and 2.4 cm on the left and right, respectively). Serum dehydroepiandrosterone levels were 6,901 ng/mL (normal range 230-2,660 ng/mL). Bilateral laparoscopic adrenalectomies were performed (left adrenalectomy first and right adrenalectomy 3 months later). Following the left adrenalectomy, the results of the endocrine evaluations were still consistent with a diagnosis of ACTH-independent Cushing's syndrome. Serum dehydroepiandrosterone sulphate levels, however, were below the normal range (143 ng/mL). Unexpectedly, the patient's menstruation resumed 2.5 months after the left adrenalectomy. Pathological examination of the resected glands showed bilateral adrenocortical adenomas, one on the left with a diameter of 3 cm, and two on the right with diameters of 0.7 cm and 1.3 cm. Immunohistochemical analysis revealed side chain cleavage, 17α-hydroxylase, 3ß-hydroxysteroid dehydrogenase, and 21-hydroxylase immunoreactivity in each adenoma. Dehydroepiandrosterone-sulfotransferase immunoreactivity was pronounced in the left adenoma, less pronounced in one of the right adenoma and weak in the other right adenoma. These results were consistent with clinical endocrine findings. Ours is the first case of a patient with bilateral cortisol-secreting adenomas with unilateral oversecretion of dehydroepiandrosterone sulphate. Resumption of menstruation after the correction of the dehydroepiandrosterone-sulphate excess, despite persistent cortisol excess, indicates the importance of adrenal androgens for the regulation of the menstrual cycle.

Characterization of steroidogenic enzyme expression in aldosterone-producing adenoma: a comparison with various human adrenal tumors.

We analyzed the expression profiles of several steroidogenic enzymes in normal adrenals, aldosterone-producing adenomas (APA), cortisol-producing adenomas combined with Cushing's syndrome (CPA) or with subclinical Cushing's syndrome (SCPA), and nonfunctioning adrenal adenomas (NFA) to clarify the nature and characteristics of steroidogenesis in APA. Clinical data were collected for all subjects. In resected adrenal glands (normal adrenals, APA, CPA, SCPA, and NFA), the mRNA expression levels of the CYP17, HSD3B2, CYP11B1, and CYP11B2 genes were studied using real-time quantitative PCR and immunohistochemistry. The CYP11B2 mRNA level in APA was significantly higher than that in other groups. The CYP17/HSD3B2 ratio for mRNA in APA was significantly lower than those in the other groups. Low ratio of CYP17/HSD3B2 with high expression of CYP11B2 seems to explain steroidogenic characteristics of APA.

Gilteritinib Monotherapy as a Transplant Bridging Option for a Patient with FLT3-Mutated Acute Promyelocytic Leukemia Who Developed a Second Relapse after All-Trans Retinoic Acid + Chemotherapy, Arsenic Trioxide, and High-Dose Cytarabine Therapy.

We report a case of FLT3-mutated APL who developed disease relapse despite all-trans retinoic acid (ATRA) + chemotherapy, and re-induction chemotherapy with arsenic trioxide (ATO) and high-dose (HD) cytarabine (Ara-C) therapy failed to yield complete remission. Because the leukemic cells were resistant to all the aforementioned therapies, we started the patient on monotherapy with gilteritinib, a selective FLT3-inhibitor, as an alternative re-induction treatment option rather than further intensive chemotherapy. The patient showed complete hematologic remission in response to this therapy. This case serves as supporting evidence for the use of single-agent therapy with gilteritinib as a bridge to transplantation in patients with refractory FLT3-mutated APL.

Ezetimibe reduces intimal hyperplasia in rabbit jugular vein graft.

BACKGROUND: The selective cholesterol transport inhibitor ezetimibe is widely used to prevent development of atherosclerosis in patients with hypercholesterolemia. However, whether this agent inhibits intimal hyperplasia in autologous vein grafts is unknown. The present study was undertaken to clarify if ezetimibe reduces cell proliferation and intimal hyperplasia in vein grafts. METHODS: Forty-four rabbits were randomly divided into two groups: one group received ezetimibe (0.6 mg/kg/d), and the control group did not. Ezetimibe administration was started 1 week before rabbits underwent interposition reversed autologous jugular vein grafts. The proliferative cells and apoptotic cells were counted in the vein grafts 14 days after implantation, and changes in acetylcholine-induced relaxation and endothelial intracellular concentration of Ca2+ ([Ca2+]i) were examined at 28 days. RESULTS: Ezetimibe reduced serum cholesterol and triglyceride. There were fewer proliferating cells in the ezetimibe group (5.7%±0.2%, n=7) than in the control group (12.8%±0.5%, n=7; P<.0001) and more apoptotic cells in the ezetimibe group (5.3%±0.2%, n=7) than in the control group (2.3%±0.2%, n=7; P<.0001). Intimal hyperplasia was less in the ezetimibe group (46.1±6.0 µm, n=7) than in the control group (76.0±2.5 µm, n=7; P<.01). Acetylcholine-produced endothelium-dependent relaxation was observed only in the ezetimibe group, which was blocked by the nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine. Acetylcholine increased [Ca2+]i only in the ezetimibe group. CONCLUSIONS: Ezetimibe reduced cell proliferation and enhanced cell apoptosis, thus inhibiting intimal hyperplasia in rabbit autologous vein grafts. Ezetimibe restored the acetylcholine-induced increase in [Ca2+]i in endothelial cells and improved endothelium-dependent NO-mediated relaxation in the vein graft. Our results suggest that ezetimibe enhances the function of endothelial NO through an increase in endothelial [Ca2+]i, thus reducing vein graft intimal hyperplasia.

Subclavian artery aneurysm in Marfan syndrome.

We present a case of a left subclavian artery aneurysm in a 48-year-old man with Marfan syndrome. Aneurysms of the subclavian artery are rare in patients with Marfan syndrome. Resection of the aneurysm and interposition with a synthetic graft were performed through a supra- and infraclavicular incision, without resecting the clavicle. Histological findings were compatible with Marfan syndrome. In patients with Marfan syndrome, regular follow-up is important because of the occurrence of peripheral aneurysms other than the aorta.

Cost disparity between open repair and endovascular aneurysm repair for abdominal aortic aneurysm: a single-institute experience in Japan.

PURPOSE: We conducted this study to compare the cost of open surgical repair (OR) with that of endovascular aneurysm repair (EVAR) of an abdominal aortic aneurysm (AAA). METHODS: Between January 2007 and November 2008, 70 patients underwent open repair and 57 patients underwent EVAR. We evaluated the total cost, including that of the Diagnosis Procedure Combination (DPC), that of the surgical procedure, that of materials such as grafts and guide wires, and that of the anesthesia. RESULTS: The mean costs for OR versus EVAR were as follows: DPC, ¥632,370 versus ¥490,050, respectively, which was significant; anesthesia, ¥123,540 versus ¥86,220, respectively (P < 0.05); and materials, ¥257,770 versus ¥2,113,280, respectively (P < 0.05). Thus, the mean total cost was ¥1,825,830 versus ¥3,159,270 for open repair and EVAR, respectively (P < 0.05). CONCLUSIONS: New technologies should not only be clinically effective, but also cost effective. EVAR is less invasive clinically, but the cost of endovascular prostheses and other materials remains high.

Intratumoral heterogeneity of steroidogenesis in aldosterone-producing adenoma revealed by intensive double- and triple-immunostaining for CYP11B2/B1 and CYP17.

INTRODUCTION: Cytochrome P450 11B2 (CYP11B2) plays a pivotal role in aldosterone synthesis, while cytochrome P450 11B1 (CYP11B1) and cytochrome P450 17A1 (CYP17) are involved in cortisol synthesis in normal human adrenal glands. However, their detailed distribution in aldosterone-producing adenoma (APA) remains incompletely settled. MATERIALS AND METHODS: We examined the status of CYP11B1/CYP11B2 and CYP11B2/CYP17A1 expressions in 27 APA (double staining) cases and 21 APA (triple staining) cases by using immunofluorescence staining and semi-quantitative evaluation. RESULTS: Tumor cells co-expressing CYP11B1/B2 (hybrid cell type A), CYP11B2/17 (hybrid cell type B), CYP11B1/17 (hybrid cell type C), and CYP11B1/B2/17 (triple-positive cell) were identified. The area and cell number of these cells were relatively small, but the size of individual hybrid cells were different between three hybrid cell types (A/B/C) and triple-positive cells. CONCLUSION: The presence of hybrid cells indicated the marked intratumoral heterogeneity of steroidogenesis in APAs, particularly in those producing glucocorticoids and mineralocorticoids.

Acute Exacerbation of Idiopathic Pulmonary Fibrosis Following Treatment for Cushing's Syndrome.

A 64-year-old Japanese man with mild reticular shadows in both lungs developed a lung tumor causing ectopic Cushing's syndrome. He was prescribed an adrenal inhibitor, which controlled his hypercortisolemia. However, he developed acute exacerbation of idiopathic pulmonary fibrosis (IPF) and died within weeks. Previous studies have suggested a dosage reduction of corticosteroids for IPF as a triggering event for acute exacerbation. The present case suggests that IPF coexisting with Cushing's syndrome may have been exacerbated after the correction of hypercortisolemia. Therefore, close monitoring of cortisol levels along with the clinical course of IPF is required in similar cases that require the correction of hypercortisolemia.

Immunohistochemical analysis of insulin-like growth factor 1 and its receptor in sporadic schwannoma/peripheral nerve sheath tumour.

OBJECTIVE: To investigate the immunohistochemical localization of insulin-like growth factor 1 (IGF-1) and IGF-1 receptor (IGF-1R) in archival specimens of sporadic schwannoma. METHOD: This study retrospectively analysed the immunolocalization of IGF-1 and IGF-1R in schwannoma specimens collected from all patients with sporadic schwannoma that were treated by two institutions in Japan. The study also evaluated the association between the extent of the IGF-1 and IGF-1R immunoreactivity and several clinicopathological characteristics (age, sex and maximum tumour dimension). RESULTS: The study examined a total of 29 sporadic schwannoma specimens. IGF-1 and IGF-1R immunoreactivity was detected in the majority of the specimens regardless of their anatomical location. IGF-1 and IGF-1R were not co-localized. There was no association between the extent of the IGF-1 and IGF-1R immunoreactivity and the clinicopathological characteristics of the patients. CONCLUSIONS: As IGF-1 and IGF-1R immunoreactivity was detected in the majority of sporadic schwannoma specimens regardless of their anatomical location, these findings suggest that an IGF-1/IGF-1R loop could play a role in the tumorigenesis and progression of schwannomas via an autocrine-paracrine mechanism.

Histopathological analysis of spontaneous large necrosis of adrenal pheochromocytoma manifested as acute attacks of alternating hypertension and hypotension: a case report.

BACKGROUND: Pheochromocytomas are rare catecholamine-producing neuroendocrine tumors. Hypertension secondary to pheochromocytoma is often paroxysmal, and patients occasionally present with sudden attacks of alternating hypertension and hypotension. Spontaneous, extensive necrosis within the tumor that is associated with catecholamine crisis is an infrequent complication of adrenal pheochromocytoma, but its pathogenesis remains unclear. CASE PRESENTATION: A 69-year-old Japanese man developed acute-onset episodic headaches, palpitations, and chest pains. During the episodes, both marked fluctuations in blood pressure (ranging from 40/25 to 300/160 mmHg) and high plasma levels of catecholamines were found simultaneously. Radiological findings indicated a 4-cm left adrenal pheochromocytoma. These episodic symptoms disappeared within 2 weeks with normalization of plasma catecholamine levels. Two months later, the patient underwent adrenalectomy. Microscopic examinations revealed pheocromocytoma with a large central area of coagulative necrosis. The necrotic material was immunohistochemically positive for chromogranin A. Granulation tissue was adjacent to the necrotic area, accompanied by numerous hemosiderin-laden macrophages and histiocytes with vascular proliferation. Viable tumor cells, detected along the periphery of the tumor, demonstrated pyknosis, and the Ki-67 labeling index was 2 % in the hot spot. No embolus or thrombus formation was found in the resected specimen harboring the whole tumor. The Pheochromocytoma of the Adrenal gland Scaled Score was 2 out of 20. The patient's postoperative course was unremarkable for > 7 years. CONCLUSIONS: Presumed causal factors for the extensive necrosis of adrenal pheochromocytoma in previously reported cases include hemorrhage into the tumor, hypotension induced by a phentolamine administration, embolic infarction, high intracapsular pressure due to malignant growth of the tumor, and catecholamine-induced vasoconstriction. In the present case, histopathological and clinical findings suggest that under conditions of chronic ischemia due to catecholamine-induced vasoconstriction, an acute infarction occurred after sudden attacks of alternating hypertension and hypotension. Over the subsequent 2 weeks, repetitive massive release of catecholamines from the infarcts into circulation likely accelerated infarction progression by causing repeated attacks of alternating hypertension and hypotension and resulted in the large necrosis. This case highlights the need for physicians to consider acute spontaneous tumor infarction accompanying episodic catecholamine crisis as a rare but severe complication of pheochromocytoma.

A Novel Mutation in the type Iα Regulatory Subunit of Protein Kinase A (PRKAR1A) in a Cushing's Syndrome Patient with Primary Pigmented Nodular Adrenocortical Disease.

A 40-year-old man presented with Cushing's syndrome due to bilateral adrenal hyperplasia with multiple nodules. Computed tomography scan results were atypical demonstrating an enlargement of the bilateral adrenal glands harboring multiple small nodules, but the lesion was clinically diagnosed to be primary pigmented nodular adrenocortical disease (PPNAD) based on both endocrinological test results and his family history. We performed bilateral adrenalectomy and confirmed the diagnosis histologically. An analysis of the patient and his mother's genomic DNA identified a novel mutation in the type Iα regulatory subunit of protein kinase A (PRKAR1A) gene; p.E17X (c.49G>T). This confirmed the diagnosis of PPNAD which is associated with Carney Complex.

3ß-Hydroxysteroid dehydrogenase isoforms in human aldosterone-producing adenoma.

It has become important to evaluate the possible involvement of 3ß-hydroxysteroid dehydrogenase type 1 (HSD3B1) and 2 (HSD3B2) isoforms in aldosterone-producing adenoma (APA). In this study, we studied 67 and 100 APA cases using real-time quantitative PCR (qPCR) and immunohistochemistry, respectively. Results of qPCR analysis demonstrated that HSD3B2 mRNA was significantly more abundant than HSD3B1 mRNA (P < 0.0001), but only HSD3B1 mRNA significantly correlated with CYP11B2 (aldosterone synthase) mRNA (P <0.0001) and plasma aldosterone concentration (PAC) of the patients (P <0.0001). Results of immunohistochemistry subsequently revealed that HSD3B2 immunoreactivity was detected in the great majority of APA but a significant correlation was also detected between HSD3B1 and CYP11B2 (P <0.0001). In KCNJ5 mutated APA, CYP11B2 mRNA (P <0.0001) and HSD3B1 mRNA (P = 0.011) were significantly higher than those of wild type APA. These results suggest that HSD3B1 is involved in aldosterone production, despite its lower levels of expression compared with HSD3B2, and also possibly associated with KCNJ5 mutation in APA.

An adverse pregnancy-associated outcome due to overlooked primary aldosteronism.

A 31-year-old woman with treatment-resistant pregnancy-induced hypertension during her first pregnancy delivered a small-for-gestational-age infant (weight: 1,070 g). After delivery, she was diagnosed with primary aldosteronism (PA) associated with a left adrenal adenoma. Following a thorough examination, she underwent laparoscopic left adrenalectomy, and the diagnosis of an aldosterone-producing adenoma was confirmed based on a pathological examination. Thereafter, the patient's hypertension and hypokalemia completely disappeared. She became pregnant again and successfully delivered her second infant at the 37th week of gestation (weight: 2,720 g) without developing treatment-resistant hypertension. Secondary causes of hypertension should not be overlooked, even in young pregnant women.

1α,25-Dihydroxyvitamin D3 inhibits the human H295R cell proliferation by cell cycle arrest: a model for a protective role of vitamin D receptor against adrenocortical cancer.

UNLABELLED: Using the human H295R adrenocortical carcinoma cell line as a model, we analyzed the role of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3)]--vitamin D receptor (VDR) axis in the growth of adrenocortical cancer (ACC). The presence of VDR in various adrenocortical tissues, including ACC, was also investigated. DNA synthesis was evaluated by [³H]thymidine cell incorporation after treatment with 1α,25(OH)2D3 at increasing doses. The effect of 1α,25(OH)2D3 on cell cycle and apoptosis was analyzed with a flow cytometer. Cyclin-dependent kinase 4 (CDK4) expression, a molecular marker of G1-S cell cycle transition phase, was evaluated in cells treated with 1α,25(OH)2D3 before and after VDR gene silencing. 1α,25(OH)2D3 treatment inhibited cell proliferation by 20% at a dose of 1 nM, in parallel with steroid secretion decrease. A cell cycle arrest in G1, with no change in apoptotic cell proportion, was observed after 10 nM 1α,25(OH)2D3 cell exposure. CDK4 activation was reduced by 10 nM 1α,25(OH)2D3 but was not affected by 1α,25(OH)2D3 after VDR gene silencing. Expression of VDR mRNA was lower in ACC than in benign adrenocortical tumors. VDR immunostaining was evident in benign tumors but it was weak in ACC tissues. CONCLUSIONS: Slightly supra-physiological concentrations of 1α,25(OH)2D3 have a moderate anti-proliferative effect on H295R cells. Anti-proliferative effect was due to cell cycle arrest in G1 phase, without inducing apoptosis. The low mRNA expression levels at qRT-PCR as well as the weak immunohistochemical expression of VDR in ACC, suggests a protective role of VDR against malignant adrenocortical growth.

Voltage-gated calcium channels in the human adrenal and primary aldosteronism.

Calcium channel blockers can efficiently be used in the treatment of primary aldosteronism (PA) related hypertension, but details on the localization of calcium channel (CC) in the human adrenal and its disorders, including PA, have remained unclear. Therefore, in this study we analyzed the known α subunits of L-, N- and T-type CCs in 74 adrenocortical aldosterone-producing adenomas (APA) and 16 cortisol-producing adenomas (CPA) using quantitative RT-PCR (qPCR). We also examined the status of L-(CaV1.2, CaV1.3), N-(CaV2.2) and T-(CaV3.2) CC subunits in five non-pathological adrenals (NA), five idiopathic hyperaldosteronism (IHA) cases, and 50 APA using immunohistochemistry. After qPCR evaluation, only CaV1.2, CaV1.3, CaV2.2, and CaV3.2 mRNA levels could be detected in APA and CPA. Among those, only CaV3.2 mRNA levels were significantly correlated with plasma aldosterone levels (P=0.0031), CYP11B2 expression levels (P<0.0001) and the presence of KCNJ5 mutations (P=0.0019) in APA. The immunolocalization of CCs in NA and IHA was detected in the zona glomerulosa (ZG), with a predominance of CaV3.2 in APA. These findings suggest that different types of CC can be involved in calcium-related aldosterone biosynthesis.

A rare case of Cushing's syndrome due to bilateral adrenocortical adenomas.

We report a rare case of Cushing's syndrome caused by bilateral cortisol-secreting adenomas in a 63-year-old man. Our preoperative diagnosis was based on endocrinological results and imaging findings. Laparoscopic adrenalectomy has become a standard technique for adrenal tumors; however, bilateral adrenalectomy results in postoperative adrenal insufficiency, necessitating lifelong steroid replacement. To preserve adrenal function, the left adrenal gland was completely resected, whereas the right adrenal gland was partially resected laparoscopically. Hydrocortisone supplementation was initiated at a dose of 30 mg/day and was slowly tapered. However, symptoms of adrenal insufficiency developed, and adrenal steroid secretion did not respond to exogenous adrenocorticotropic hormone. Bilateral cortisol-secreting tumors rarely cause Cushing's syndrome. The present study comprised few patients, and the utilized surgical procedures (i.e., total/partial adrenalectomy or bilateral total adrenalectomy) were not uniform. Few cases of bilateral adrenal-preserving surgery have been reported. However, our patient developed adrenal insufficiency after the oral cortisone supplementation was tapered. This report demonstrates that partial adrenalectomy does not necessarily preserve normal adrenocortical function. Therefore, careful postoperative observation is necessary for patients undergoing a partial adrenalectomy.

Development of monoclonal antibodies against human CYP11B1 and CYP11B2.

1. The final enzymes in the biosynthesis of aldosterone and cortisol are by the cytochrome P450 CYP11B2 and CYP11B1, respectively. The enzymes are 93% homologous at the amino acid level and specific antibodies have been difficult to generate. 2. Mice and rats were immunized with multiple peptides conjugated to various immunogenic proteins and monoclonal antibodies were generated. The only peptide sequences that generated specific antibodies were amino acids 41-52 for the CYP11B2 and amino acids 80-90 for the CYP11B1 enzyme. 3. The mouse monoclonal CYP11B2-41 was specific and sensitive for use in western blots and produced specific staining of the zona glomerulosa of normal adrenal glands. The rat monoclonal CYP11B1-80 also detected a single band by western blot and detected only the zona fasciculata. Triple immunofluorescence of the adrenal demonstrated that the CYP11B1 and the CYP11B2 did not co-localize, while as expected the CYP11B1 co-localized with the 17α-hydroxylase.