Evaluation of cell proliferation in rat tissues with BrdU, PCNA, Ki-67(MIB-5) immunohistochemistry and in situ hybridization for histone mRNA.

The standard method for assessment of cell proliferation in paraffin-embedded tissue sections is 5-bromodeoxyuridine (BrdU) immunohistochemistry (IHC). BrdU can be administered to laboratory animals via IP injections, is readily incorporated into nuclei during the DNA synthetic phase of the cell cycle, and is detected with an anti-BrdU antibody. This method has several disadvantages, and an accurate method for evaluation of proliferative activity that can substitute for BrdU IHC, when necessary, is of great interest to investigators. Alternative methods for detection of proliferating cells in tissue sections are proliferating cell nuclear antigen (PCNA) IHC, Ki-67 IHC, and in situ hybridization (ISH) for histone mRNA. To determine the optimal choice, we analyzed the correlation of anti-PCNA, anti-Ki-67(MIB-5), and histone mRNA labeling indices (LIs) with anti-BrdU LI in rat highly replicative (renewing) tissues. The correlation between anti-BrdU and histone mRNA LIs, as well as the correlation between anti-BrdU and anti-Ki-67 LIs, was statistically significant. There was no significant correlation between anti-BrdU and anti-PCNA LIs. These results suggest that both ISH for histone mRNA and IHC with MIB-5 are preferable techniques for assessment of cell proliferation in rat paraffin-embedded renewing tissues compared to PCNA IHC. They can substitute for BrdU IHC when necessary.     

Multiparameter analysis of AgNOR in thyroid lesions: comparison with PCNA expression

The aim of the study was to examine numerous features of argyrophilic proteins related to nucleolar organizer regions (AgNORs) in thyroid tumors, relate them to PCNA expression and evaluate which of these features might be useful in the diagnosis of thyroid lesions. Paraffin sections of 100 thyroid tumors were silver-stained and divided into 9 groups: nodular goiter (NG), simple adenoma (SA), microfollicular adenoma (MFA), follicular carcinoma (FC), follicular variant of papillary carcinoma (PC-F), classical variant of papillary carcinoma (PC-C), Hürthle cell adenoma (HA), Hürthle cell carcinoma (HC), and anaplastic carcinoma (AC). The slides were analyzed with the computerized system for image analysis. A weak correlation was found between PCNA expression and AgNOR size. AC differed significantly from all other examined groups in many features of AgNOR dots. Hürthle cell neoplasms were characterized by the presence of a usually single and relatively large dot. With respect to diagnosing follicular lesions, we found that the evaluation of the total area of dots in the nucleus seemed to be the most useful for discrimination: the assumption of 4.9 micro m2, as a cut-off value, allowed a correct classification of 77% of FC cases. Computer-aided morphometric analysis of AgNORs may be useful in the diagnostics of thyroid lesions.     

Epithelial cell proliferative activity and oral cancer progression

Accurate, predictive assessment of the behaviour and progression of oral cancers and precancers remains elusive in clinical practice. Archival tissue specimens from 10 previously treated patients with oral lesions of known clinical outcome (3 years post-treatment) were re-examined histopathologically, and proliferative cell labelling indices (LIs) determined for Ki67, cyclin A and histone mRNA cell cycle markers. While histone mRNA labelling ultimately proved unreliable, both Ki67 and cyclin A LIs demonstrated a clear trend for enhanced labelling to occur in increasingly dysplastic and neoplastic tissue, with particular emphasis on suprabasal labelling in abnormal tissue. Perhaps of greatest significance was the observation of increased LIs and suprabasal labelling in lesions with poor clinical outcome, such as patients developing recurrent disease or cervical lymph node metastasis. Measurement of cell proliferative activity in individual oral epithelial dysplastic lesions or invasive squamous cell carcinomas may thus provide unique, predictive information on clinical outcome.     

Effects of 39 Compounds on Calmodulin-Regulated Adenylyl Cyclases AC1 and Bacillus anthracis Edema Factor

Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP. Membranous AC1 (AC1) is involved in processes of memory and learning and in muscle pain. The AC toxin edema factor (EF) of Bacillus anthracis is involved in the development of anthrax. Both ACs are stimulated by the eukaryotic Ca²⁺-sensor calmodulin (CaM). The CaM-AC interaction could constitute a potential target to enhance or impair the AC activity of AC1 and EF to intervene in above (patho)physiological mechanisms. Thus, we analyzed the impact of 39 compounds including typical CaM-inhibitors, an anticonvulsant, an anticholinergic, antidepressants, antipsychotics and Ca²⁺-antagonists on CaM-stimulated catalytic activity of AC1 and EF. Compounds were tested at 10 μM, i.e., a concentration that can be reached therapeutically for certain antidepressants and antipsychotics. Calmidazolium chloride decreased CaM-stimulated AC1 activity moderately by about 30%. In contrast, CaM-stimulated EF activity was abrogated by calmidazolium chloride and additionally decreased by chlorpromazine, felodipine, penfluridol and trifluoperazine by about 20–40%. The activity of both ACs was decreased by calmidazolium chloride in the presence and absence of CaM. Thus, CaM-stimulated AC1 activity is more insensitive to inhibition by small molecules than CaM-stimulated EF activity. Inhibition of AC1 and EF by calmidazolium chloride is largely mediated via a CaM-independent allosteric mechanism.     

Interactions of acyl carnitines with model membranes: a (13)C-NMR study

Esterification of fatty acids with the small polar molecule carnitine is a required step for the regulated flow of fatty acids into mitochondrial inner matrix. We have studied the interactions of acyl carnitines (ACs) with model membranes [egg yolk phosphatidylcholine (PC) vesicles] by (13)C-nuclear magnetic resonance (NMR) spectroscopy. Using AC with (13)C-enrichment of the carbonyl carbon of the acyl chain, we detected NMR signals from AC on the inside and outside leaflets of the bilayer of small unilamellar vesicles prepared by cosonication of PC and AC. However, when AC was added to the outside of pre-formed PC vesicles, only the signal for AC bound to the outer leaflet was observed, even after hours at equilibrium. The extremely slow transmembrane diffusion ("flip-flop") is consistent with the zwitterionic nature of the carnitine head group and the known requirement of transport proteins for movement of ACs through the mitochondrial membrane. The partitioning of ACs (8-18 carbons) between water and PC vesicles was studied by monitoring the [(13)C]carbonyl chemical shift of ACs as a function of pH and concentration of vesicles. Significant partitioning into the water phase was detected for ACs with chain lengths of 12 carbons or less. The effect of ACs on the integrity of the bilayer was examined in vesicles with up to 25 mol% myristoyl carnitine; no gross disruption of the bilayer was observed. We hypothesize that the effects of high levels of long-chain AC (as found in ischemia or in certain diseases) on cell membranes result from molecular effects on membrane functions rather than from gross disruption of the lipid bilayer.     

Characterization of GFAP expression and cell proliferation in the rat median eminence following hypophysectomy

To analyze whether the reorganization of the rat median eminence after hypophysectomy might be related to changes in glial fibrillary acidic protein (GFAP)- and cellular proliferation, the distribution of cells immunoreactive for GFAP and the proliferation rate of such cells were analyzed at 20, 40 and 60 days posthypophysectomy. For this study, four rostro-caudal regions of the median eminence were differentiated: the retrochiasmatic, preinfundibular, infundibular and postinfundibular regions. In each of these regions, three layers were studied: the ependymal, the internal and the external. At 20 and 40 days after hypophysectomy, significant increases in cellular proliferation affecting all three layers studied in the preinfundibular and infundibular regions were found. At the same time points, increases in GFAP expression were also observed. However, after 60 days, GFAP and proliferative cellular nuclear antigen (PCNA) expression decreased. Although variations of PCNA and GFAP levels were evident, no colocalisation of PCNA and GFAP was found in the cells of the median eminence in untreated or hypophysectomized rats when sections were analyzed by double immunohistochemical staining. Our results suggest that reorganization of median eminence involves alterations (or modulation) of GFAP-immunoreactive cells together with a proliferation of cells that are not GFAP-immunoreactive. This study also demonstrates that this reorganization is completed within the first two months after hypophysectomy.     

Repeated acidosis challenges and live yeast supplementation shape rumen microbiota and fermentations and modulate inflammatory status in sheep

This study aimed to investigate the impact of repeated acidosis challenges (ACs) and the effect of live yeast supplementation (Saccharomyces cerevisiae I-1077, SC) on rumen fermentation, microbial ecosystem and inflammatory response. The experimental design involved two groups (SC, n=6; Control, n=6) of rumen fistulated wethers that were successively exposed to three ACs of 5 days each, preceded and followed by resting periods (RPs) of 23 days. AC diets consisted of 60% wheat-based concentrate and 40% hay, whereas RPs diets consisted of 20% concentrate and 80% hay. ACs induced changes in rumen fermentative parameters (pH, lactate and volatile fatty-acid concentrations and proportions) as well as in microbiota composition and diversity. The first challenge drove the fermentation pattern towards propionate. During successive challenges, rumen pH measures worsened in the control group and the fermentation profile was characterised by a higher butyrate proportion and changes in the microbiota. The first AC induced a strong release of rumen histamine and lipopolysaccharide that triggered the increase of acute-phase proteins in the plasma. This inflammatory status was maintained during all AC repetitions. Our study suggests that the response of sheep to an acidosis diet is greatly influenced by the feeding history of individuals. In live yeast-supplemented animals, the first AC was as drastic as in control sheep. However, during subsequent challenges, yeast supplementation contributed to stabilise fermentative parameters, promoted protozoal numbers and decreased lactate producing bacteria. At the systemic level, yeast helped normalising the inflammatory status of the animals.     

Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides

Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes. These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl(2). N-Methylanthraniloyl (MANT)-GTP inhibited C1.C2 with a K(i) of 4.2 nm. Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group. MANT-inosine 5'-[gamma-thio]triphosphate potently inhibited C1.C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins. Inosine 5'-[gamma-thio]triphosphate and uridine 5'-[gamma-thio]triphosphate were mixed G-protein activators and AC inhibitors. AC5 was up to 15-fold more sensitive to inhibitors than AC2. EF and ACT exhibited unique inhibitor profiles. At sAC, 2',5'-dideoxyadenosine 3'-triphosphate was the most potent compound (IC(50), 690 nm). Several MANT-adenine and MANT-guanine nucleotides inhibited sGC with K(i) values in the 200-400 nm range. UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors. The exchange of MnCl(2) against MgCl(2) reduced inhibitor potencies at ACs and sGC 1.5-250-fold, depending on the nucleotide and cyclase studied. The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase. Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.     

Cellular autophagic capacity changes during azaserine-induced tumour progression in the rat pancreas. Up-regulation in all premalignant stages and down-regulation with loss of cycloheximide sensitivity of segregation along with malignant transformation

The knowledge of alterations in regulation of autophagy during tumorigenesis may also help our understanding of its normal control. We established an experimental system and reported recently that autophagic capacity, measured as the cell's capability of increasing segregation (formation of autophagosomes) and subsequent degradation of cytoplasmic quanta were highly increased in premalignant nodule cells 6 months after initiation by azaserine in the rat pancreas in vivo. In the present study, we followed changes of these autophagic functions throughout the tumour progression. We carried out electron-microscopic morphometrical analysis of the expansion of autophagic vacuole compartment and subcompartments induced by vinblastine (an in vivo segregation enhancer), as well as their regression upon segregation-inhibitor cycloheximide post-treatment. Premalignant tumour samples were taken at month 5, month 8 (nodules), month 10 and month 15 (adenomas) after initiation. In all these stages, a highly increased and varying autophagic capacity was found compared with the host tissue. The basal (non-stimulated) autophagic compartment was measurable only at month 5 and month 15, and its regression upon cycloheximide was consistent with increased basal autophagic activity. Compared with the host tissue, autophagic capacity profoundly decreased in the differentiated and anaplastic adenocarcinomas at month 20, when, surprisingly, cycloheximide was unable to inhibit segregation. Our conclusion is that down-regulation of the cycloheximide sensitive segregation and a partly compensatory up-regulation of an alternative pathway of segregation might occur along with malignant transformation.     

Natural regulatory cells in murine bone marrow: inhibition of in vitro proliferative and cytotoxic responses to alloantigens

A lymphocyte-enriched fraction of murine bone marrow (BML), obtained by sucrose density fractionation, contains natural regulatory cells that can profoundly suppress the proliferative and cytotoxic response of syngeneic lymph node cells to irradiated alloantigens in a mixed lymphocyte culture (MLC). A close correlation exists between the inhibition of alloantigen-induced proliferation and the generation of cytotoxic effectors. The suppression of proliferation is dependent on the dose of BML added to the cultures but is not due to cell crowding, since red blood cells, thymocytes, and irradiated splenocytes, all syngeneic to the lymph node responders, do not inhibit proliferation to the degree observed with BML. The addition of BML to cultures does not cause the maximum proliferative response to change from the usual day 5 peak, indicating that there is no change in culture kinetics. The release of nonradioactive thymidine by BML cannot explain the suppression. The target of suppression is maximally affected during the first 24 hr of culture, since adding BML to MLC later than this resulted in negligible inhibition of proliferation. Thus, the natural regulatory cell-mediated suppression reflects inhibition of "early" events in the proliferative response to alloantigens.     

Equine fetal kinetics: Presentation and location

The extent and nature of fetal mobility (presentation and location changes) were studied in 10 pony mares by ultrasonic examinations each week during the fetal stage (Day 40 to term). The percentage of examinations with cranial fetal presentation was 35 to 43% during Months 2 to 5 (no significant differences among months); a significant increase occurred between Months 5 and 6 reaching 100% at Month 7. With only 3 transient exceptions, the fetus was in cranial presentation during all examinations from Month 7 to term. The frequency of presentation changes between successive examinations decreased (P<0.05) between Months 4 and 5 and further decreased between Months 6 and 7. A significant and progressive decrease in the proportion of examinations in which at least part of the fetus was located in the umbilical-cord horn occurred over Months 2 to 4. During the same months, an increase occurred in the frequency of location of at least part of the fetus in the noncord horn and in the frequency of location of all of the fetus in the uterine body. The fetus was equally distributed among the 3 parts of the uterus during Month 4. Thereafter, locations decreased (P<0.05) in the noncord horn, increased (P<0.05) in the uterine body, and maintained a plateau (no significant differences) in the cord horn until Month 7 and then increased (P<0.05). The frequency of location changes between successive examinations increased (P<0.01) progressively during Months 2 to 4 followed by a decrease between Months 4 and 10. With one exception, the fetus was located in both the cord horn and uterine body for all examinations during Month 9 to term. In conclusion, a gradual decrease in fetal mobility occurred after Month 4; with only a few transient exceptions, final selection of cranial presentation occurred by Month 7, and final selection of partial location in the cord horn occurred by Month 9.     

Changes of colon epithelium proliferation due to individual aging with cyclin proliferating cell nuclear antigen (PCNA/cyclin) immunostaining compared to [3H]-thymidine radioautography

We report a change in the proliferative activity of mouse colonic epithelium due to development and aging. In order to measure the proliferative activity, colonic epithelium was immunostained for cyclin proliferating cell nuclear antigen (PCNA/cyclin), which appears from the Gl to the S phase of the cell cycle, and compared with labeling obtained by [³H]-thymidine radioautography. Litter mice of six age groups from the fetal period (embryonic day 19), newborn period (postnatal day 1), suckling period (postnatal day 5), weaning period (postnatal dy 21), adult period (2 month old) to the senescent period (11 month old) were examined by immunohistochemistry. The descending colons were fixed in methacarn (method-Carnoy) and embedded in paraffin. Sections were stained for PCNA/cyclin activity using 19A2 monoclonal antibody and the avidin-biotin peroxidase complex (ABC) technique. For radioautography, litter mice of nine age groups using in vivo intraperitoneal administration of [³H]-thymidine. The labeling indices of colonic epithelial cells in the proliferative zone were then analyzed and compared between the two investigative methods. Our results show that the prliferative activity of mice colon was high in the fetal and newborn periods and almost constant from the suckling period to senescence, as demonstrated by both PCNA/cyclin immunohistochemistry and [³H]-thymidine radioautography. The labeling index seen by PCNA/cyclin immunohistochemistry was, however, higher than that seen by [³H]-thymidine radioautography.     

Relation of proliferative activity to survival in patients with advanced gastric cancer

The proliferative activities of 98 biopsied specimens of advanced gastric cancers were measured by the in vitro bromodeoxyuridine (BrdU) labelling method. BrdU labelling indices (LIs) varied from 4.8 to 45.1%. There was no correlation of BrdU LIs and histological type, distant metastasis, serosal invasion, macroscopic type or tumor size. However, BrdU LIs of tumors with lymph node metastases were significantly higher than those without them. Patients with tumors showing high BrdU LIs had a three-fold relative risk of death, as compared with those showing low BrdU LIs. When the BRdU LIs and all the clinicopathological parameters were entered simultaneously into the Cox model, BrdU LIs, peritoneal dissemination and serosal invasion emerged as independent prognostic parameters. These results indicate that the in vitro BrdU labelling method using biopsied materials may be useful in predicting lymph node metastasis preoperatively and designing operative procedures for individual patients.     

Gsα stimulation of mammalian adenylate cyclases regulated by their hexahelical membrane anchors

Mammalian adenylate cyclases (ACs) are pseudoheterodimers with dissimilar hexahelical membrane-anchors, isoform-specifically conserved for more than half a billion years. We exchanged both membrane anchors of the AC isoform 2 by the quorum-sensing receptor from Vibrio harveyi, CqsS, which has a ligand, Cholera-Autoinducer-1 (CAI-1). In the chimera, AC activity was stimulated by Gsα, CAI-1 had no effect. Surprisingly, CAI-1 inhibited Gsα stimulation. We report that Gsα stimulation of human AC isoforms 2, 3, 5, and 9 expressed in Sf9 cells is inhibited by serum as is AC activity in membranes isolated from rat brain cortex. AC2 activation by forskolin or forskolin/Gsα was similarly inhibited. Obviously, serum contains as yet unidentified factors affecting AC activity. The data establish a linkage in ACs, in which the membrane anchors, as receptors, transduce extracellular signals to the cytosolic catalytic dimer. A mechanistic three state model of AC regulation is presented compatible with all known regulatory inputs into mammalian ACs. The data allow designating the membrane anchors of mammalian ACs as orphan receptors, and establish a new level of AC regulation.     

Proliferating cell nuclear antigen (PCNA) expression in pituitary adenomas: relationship to the endocrine phenotype of adenoma

The expression of proliferating cell nuclear antigen (PCNA) correlates to cell proliferation and for this reason it is commonly considered as one of proliferation markers. Since proliferation rate is an important factor determining the tumor aggressiveness, the evaluation of PCNA index (the percentage of PCNA-immunopositive nuclei in the investigated tumor sample) is suggested as useful in predicting pituitary adenoma outcome. Seventy three unselected, surgically removed pituitary adenomas were immunostained with antibodies against the pituitary hormones or their subunits and against the proliferating cell nuclear antigen (PCNA). The highest PCNA index was found in ACTH-immunopositive tumors without the manifestation of the Cushing's disease ("silent" corticotropinomas). This value was significantly different in comparison to other adenoma subtypes including corticotropinomas manifesting themselves by Cushing's disease. The lowest PCNA index was noticed in monohormonal GH-secreting tumors. The adenomas which express more than one hormone (plurihormonal adenomas) seem to have a higher PCNA indices than monohormonal ones; the difference was significant in the case of mono- and plurihormonal prolactinomas. The recurrent tumors presented a higher mean PCNA index as compared to the primary tumors, although the difference was significant only in the case of prolactinomas. These findings suggest that the proliferative potential of pituitary adenomas is related to the tumor recurrence and hormone expression.     

Down regulation of ribosomal protein mRNAs during neuronal differentiation of human NTERA2 cells

We have analysed the expression of 32 ribosomal protein (RP) mRNAs during retinoic acid induced neuronal differentiation of human NTERA2 cells. Except for a new S27 variant (S27v), all were down regulated both in selectively replated differentiated neurons and the most differentiated continuous cultures, i.e., non-replated cultures. However, the expression profiles of the individual RP mRNAs were different, most (L3, L7, L8, L10, L13, L23a, L27a, L36a, L39, P0, S2, S3, S3a, S4X, S6, S9, S12, S13, S16, S19, S20, S23, and S27a) exhibited a constant down regulation, whereas a few were either initially constant (L11, L32, S8, and S11) or up regulated (L6, L15, L17, L31, and S27y) and then down regulated. The expression of S27v remained elevated in the most differentiated continuous cultures but was down regulated in replated differentiated neurons. The down regulation of RP mRNAs was variable: the expression levels in differentiated replated neurons were between 10% (S3) and 90% (S11) of the levels in undifferentiated cells. The ratio between rRNA and RP mRNA changed during the differentiation; in differentiated neurons there were, on average, about half the number of RP mRNAs per rRNA as compared to undifferentiated cells. The expression profiles of a few translation-related proteins were also determined. EF1alpha1, EF1beta1, and EF1delta were down regulated, whereas the expression of the neuron and muscle specific EF1alpha2 increased. The reduction in the expression of RP mRNAs was coordinated with a reduction in the expression level of the proliferation marker PCNA. The expression levels of most RP mRNAs were lower in purified differentiated post-mitotic neurons than in the most differentiated continuous cultures, despite similar levels of PCNA, suggesting that both the differentiation state and the proliferative status of the cells affect the expression of RP mRNAs.     

Introducing a new category of activity cliffs with chemical modifications at multiple sites and rationalizing contributions of individual substitutions

Activity cliffs (ACs) are formed by structurally similar active compounds with large potency differences. In medicinal chemistry, ACs are of high interest because they reveal structure-activity relationship (SAR) information and SAR determinants. Herein, we introduce a new type of ACs that consist of analog pairs with different substitutions at multiple sites (multi-site ACs; msACs). A systematic search for msACs across different classes of bioactive compounds identified more than 4000 of such ACs, most of which had substitutions at two sites (dual-site ACs; dsACs). A hierarchical analog data structure was designed to analyze contributions of individual substitutions to AC formation. Single substitutions were frequently found to determine potency differences captured by dsACs. Hence, in such cases, there was redundancy of AC information. In instances where both substitutions made significant contributions to dsACs, additive, synergistic, and compensatory effects were observed. Taken together, the results of our analysis revealed the prevalence of single-site ACs (ssACs) in analog series, followed by dsACs, which reveal different ways in which paired substitutions contribute to the formation of ACs and modulate SARs.     

Significant Hypercortisolism During Fractionated Radiotherapy in a Patient with a Large Corticotroph Adenoma: A Case Report and Literature Review

ObjectiveWe describe a patient with a large, invasive corticotroph adenoma who developed severe hypercortisolism shortly after starting fractionated radiotherapy.MethodsWe reviewed the patient’s clinical course, along with relevant literature for similar reported cases.ResultsA 29-year-old man was referred for radiotherapy for a residual and recurrent, invasive corticotroph adenoma. Prior to radiotherapy, he had a normal urine free cortisol (UFC) level of 44.7 μg/24 hours, with minimal symptoms. Within 2 weeks of radiotherapy, he developed hypertension, ankle edema, and hypokalemia (potassium level, 2.8 mEq/L), with a markedly elevated UFC level of 9,203 μg/24 hours. His UFC gradually decreased and normalized by the end of radiotherapy. One month later, the patient became adrenal insufficient, with a nondetectable 24-hour UFC. His adrenal function slowly recovered in 3 months. We are aware of only one previous case report of clinically significant hypercortisolism following radiotherapy in Cushing disease.ConclusionRadiotherapy may result in acute severe hypercortisolism in patients with a large corticotroph adenoma. This uncommon, but clinically significant, acute adverse effect of radiotherapy suggests that clinical observation and biochemical monitoring during or soon after radiotherapy may be indicated. (Endocr Pract. 2014;20:e166-e170)     

Cathepsin E Is a Marker of Gastric Differentiation and Signet-Ring Cell Carcinoma of Stomach: A Novel Suggestion on Gastric Tumorigenesis

Gastric cancer (GC) presents various histological features, though the mechanism underlying its diversity is seldom elucidated. It is mainly classified into well differentiated tubular adenocarcinoma (tub1), moderately differentiated tubular adenocarcinoma (tub2), poorly differentiated adenocarcinoma (por), signet-ring cell carcinoma (sig), mucinous adenocarcinoma (muc), and papillary adenocarcinoma (pap). By screening, we found cathepsin E (CTSE) expresses universally in sig-type, occasionally in por-type, and rarely in tub1/tub2-type GC cell lines. In surgically-resected specimens, CTSE was immunostained in 50/51 sig-type (98.0%), 3/10 tub1-type (30.0%), 7/18 tub2-type (38.9%), 15/26 por-type (57.7%), 4/10 pap-type (40.0%), and 0/3 muc-type (0.0%) GC. In endoscopically-resected specimens, 6/7 sig-type (85.7%), 7/52 tub1-type (13.7%), 5/12 tub2-type (41.7%), 2/7 pap-type (28.6%) GC and 0/6 adenoma (0.0%) expressed CTSE. For non-malignant tissues, CTSE is universally expressed in normal fundic, pyloric, and cardiac glands of stomach, but hardly in other digestive organs. In the precancerous intestinal metaplasia of stomach, CTSE is mostly observed in mixed gastric-and-intestinal type and deficient in solely-intestinal type. CTSE expression is positively correlated with gastric marker MUC5AC (p<0.0001) and negatively correlated with intestinal marker MUC2 (p = 0.0019). For sig-type GC, in both tumors and background mucosa, expression of MUC5AC and CTSE is high whereas that of MUC2 is low, indicating that sig-type GC reflects the features of background mucosa. For gastric adenoma and tub1/tub2-type GC, more undifferentiated tumors tend to show higher expression of CTSE with MUC5AC and lower expression of MUC2 in tumors, but they tend to present lower expression of CTSE, MUC5AC and MUC2 in background mucosa. These suggest that more malignant gastric adenocarcinoma with stronger gastric and weaker intestinal properties tend to arise from background mucosa with decreased both gastric and intestinal features. In conclusion, CTSE is a marker of both gastric differentiation and signet-ring cell carcinoma, which should shed light on the mechanism of gastric tumorigenesis.