Notch inhibition counteracts Paneth cell death in absence of caspase-8.

Opposing activities of Notch and Wnt signaling regulate mucosal barrier homeostasis and differentiation of intestinal epithelial cells. Specifically, Wnt activity is essential for differentiation of secretory cells including Wnt3-producing Paneth cells, whereas Notch signaling strongly promotes generation of absorptive cells. Loss of caspase-8 in intestinal epithelium (casp8∆int) is associated with fulminant epithelial necroptosis, severe Paneth cell death, secondary intestinal inflammation, and an increase in Notch activity. Here, we found that pharmacological Notch inhibition with dibenzazepine (DBZ) is able to essentially rescue the loss of Paneth cells, deescalate the inflammatory phenotype, and reduce intestinal permeability in casp8∆int mice. The secretory cell metaplasia in DBZ-treated casp8∆int animals is proliferative, indicating for Notch activities partially insensitive to gamma-secretase inhibition in a casp8∆int background. Our data suggest that casp8 acts in the intestinal Notch network.

Salivary MMP-9 in the detection of oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most common malignant tumour of the oral cavity. Detection of OSCC is currently based on clinical oral examination combined with histopathological evaluation of a biopsy sample. Direct contact between saliva and the oral cancer makes measurement of salivary metalloproteinase-9 (MMP-9) an attractive alternative. MATERIAL AND METHODS: In total, 30 OSCC patients and 30 healthy controls were included in this prospective study. Saliva samples from both groups were collected, centrifuged and supernatant fluid was subjected to ELISA for assessment of MMP-9. The median salivary MMP-9 values with interquartile range (IQR) of OSCC patients and the control group were statistically analysed using the Mann-Whitney U-test. The receiver operating characteristic (ROC) curve was constructed and the area under curve (AUC) was computed. RESULTS: The median absorbance MMP-9 value of the OSCC group was 0.186 (IQR= 0.158) and that of control group was 0.156 (IQR=0.102). MMP-9 was significantly increased in the OSCC patients than in the controls by +19.2% (p=0.008). Median values in patients with recurrence and in patients with primary event were 0.233 (IQR=0.299) and 0.186 (IQR=0.134) respectively. MMP-9 was significantly increased in patients with primary event (p=0.017) compared to controls by +19.2%. No significant increase of MMP-9 level was detected when comparing patients with recurrence and healthy controls (+49.4%; p=0.074). The sensitivity value of MMP-9 was 100% whereas the specificity value was 26.7% with AUC of 0.698. CONCLUSIONS: The present data indicates that the elevation of salivary levels of MMP-9 may be a useful adjunctive diagnostic tool for detection of OSCC. However, further studies are necessary to provide scientific and clinical validation.

Intercalated Cell Depletion and Vacuolar H+-ATPase Mistargeting in an Ae1 R607H Knockin Model.

Distal nephron acid secretion is mediated by highly specialized type A intercalated cells (A-ICs), which contain vacuolar H+-ATPase (V-type ATPase)-rich vesicles that fuse with the apical plasma membrane on demand. Intracellular bicarbonate generated by luminal H+ secretion is removed by the basolateral anion-exchanger AE1. Chronically reduced renal acid excretion in distal renal tubular acidosis (dRTA) may lead to nephrocalcinosis and renal failure. Studies in MDCK monolayers led to the proposal of a dominant-negative trafficking mechanism to explain AE1-associated dominant dRTA. To test this hypothesis in vivo, we generated an Ae1 R607H knockin mouse, which corresponds to the most common dominant dRTA mutation in human AE1, R589H. Compared with wild-type mice, heterozygous and homozygous R607H knockin mice displayed incomplete dRTA characterized by compensatory upregulation of the Na+/HCO3- cotransporter NBCn1. Red blood cell Ae1-mediated anion-exchange activity and surface polypeptide expression did not change. Mutant mice expressed far less Ae1 in A-ICs, but basolateral targeting of the mutant protein was preserved. Notably, mutant mice also exhibited reduced expression of V-type ATPase and compromised targeting of this proton pump to the plasma membrane upon acid challenge. Accumulation of p62- and ubiquitin-positive material in A-ICs of knockin mice suggested a defect in the degradative pathway, which may explain the observed loss of A-ICs. R607H knockin did not affect type B intercalated cells. We propose that reduced basolateral anion-exchange activity in A-ICs inhibits trafficking and regulation of V-type ATPase, compromising luminal H+ secretion and possibly lysosomal acidification.

11C-metomidate positron emission tomography after dexamethasone suppression for detection of small adrenocortical adenomas in primary aldosteronism.

PURPOSE: To evaluate whether dexamethasone suppression treatment can improve (11) C-metomidate positron emission tomography (MTO-PET) detection of small adrenocortical adenomas in primary aldosteronism (PA). MATERIALS AND METHODS: Eleven patients with proven PA and two patients with non-hyperfunctioning adrenocortical incidentalomas and small adrenocortical tumours observed on CT underwent MTO-PET before and 3 days after administration of oral dexamethasone suppression treatment. Small "hot-spot" regions of interest comprising 4 pixels (SUVhs) and 1 pixel (SUVmax) were placed in the tumour area with the highest radioactivity concentration and their respective standardised uptake values (SUV) were recorded. RESULTS: All tumours were detected and categorised as adrenocortical by MTO-PET. SUVhs as well as SUVmax were higher in PA compared to nonfunctional adenomas. Normal adrenal cortex was suppressed after dexamethasone (p < 0.05), but tumour SUV was not significantly decreased after suppression in either PA or nonfunctional tumours (p > 0.05). However, these changes caused no significant increase in the tumour-to-normal adrenal ratio (p > 0.05). CONCLUSION: MTO-PET is a highly sensitive method for detecting and categorising even small adrenocortical tumours in PA. In this series, dexamethasone-suppressed MTO-PET was unable to increase the tumour-to-normal adrenal ratio to further facilitate detection of small adenomas in PA as an alternative to adrenal venous sampling.

Identification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist.

The porcine reproductive and respiratory syndrome virus nsp1 is predicted to be auto-cleaved from the replicase polyprotein into nsp1alpha and nsp1beta subunits. In infected cells, we detected the actual existence of nsp1alpha and nsp1beta. Cleavage sites between nsp1alpha/nsp1beta and nsp1beta/nsp2 were identified by protein microsequencing analysis. Time course study showed that nsp1alpha and nsp1beta mainly localize into the cell nucleus after 10 h post infection. Further analysis revealed that both proteins dramatically inhibited IFN-beta expression. The nsp1beta was observed to significantly inhibit expression from an interferon-stimulated response element promoter after Sendai virus infection or interferon treatment. It was further determined to inhibit nuclear translocation of STAT1 in the JAK-STAT signaling pathway. These results demonstrated that nsp1beta has ability to inhibit both interferon synthesis and signaling, while nsp1alpha alone strongly inhibits interferon synthesis. These findings provide important insights into mechanisms of nsp1 in PRRSV pathogenesis and its impact in vaccine development.

Long-term effects of surgical correction of adrenal hyperplasia and adenoma causing primary aldosteronism.

PURPOSE: The purpose of this is to study long-time results of surgery for primary aldosteronism. MATERIALS AND METHODS: Thirty patients operated on for primary aldosteronism were followed for an average of 7 years. All but five required potassium substitution. Systolic as well as diastolic hypertension (mean 157/93 mmHg) was present necessitating one to five antihypertensive drugs daily (mean 2.33). Preoperative indications for surgery included presumed adenoma (aldosterone-producing adenoma (APA)) or in one case unilateral dominance of hyperplasia. RESULTS: Histopathology was classified into adenoma (n = 9), dominant nodule (n = 16), and general hyperplasia without dominating nodules (n = 5), demonstrating a higher frequency of hyperplasia than anticipated. Long-term results revealed well-controlled blood pressure (BP; mean 134/80 mmHg). Antihypertensive medication was reduced (average of 1.78 per day), but only 36% of the patients were taken off these drugs completely. S-Aldosterone was normalized. All but one (a recurrence) were normokalemic without potassium substitution at follow-up. The APA group needed less medication (median 0.5 vs. 1.5 and 2 per day) and more patients in this group were totally medication free (50%). Two recurrences occurred in the group with general hyperplasia without dominating nodules. CONCLUSION: Nodular hyperplasia is more common than anticipated. Hypersecretion of aldosterone may be released from a large nodule identified as an adenoma, as well as from a generally hyperplastic gland that has not been identified as such. Nevertheless, surgery for lateralized disease results in good long-term control of BP with less antihypertensive medication. However, patients with dominant nodule or general hyperplasia without dominating nodules need more postoperative treatment than patients with APA. The majority of patients do not achieve normotension without medications, but they do become normokalemic.

Mutations in FAM134B, encoding a newly identified Golgi protein, cause severe sensory and autonomic neuropathy.

Hereditary sensory and autonomic neuropathy type II (HSAN II) leads to severe mutilations because of impaired nociception and autonomic dysfunction. Here we show that loss-of-function mutations in FAM134B, encoding a newly identified cis-Golgi protein, cause HSAN II. Fam134b knockdown results in structural alterations of the cis-Golgi compartment and induces apoptosis in some primary dorsal root ganglion neurons. This implicates FAM134B as critical in long-term survival of nociceptive and autonomic ganglion neurons.

Adrenocorticotropin-independent Cushing's syndrome in pregnancy related to overexpression of adrenal luteinizing hormone/human chorionic gonadotropin receptors.

Cushing's syndrome during pregnancy is rare, and rather than being of pituitary origin most patients exhibit ACTH-independent adrenal hypercortisolism. In some cases the syndrome has spontaneously resolved post partum, suggesting the presence of a pregnancy-associated stimulatory factor(s). We describe a case with aberrant adrenal LH/hCG receptors in a large adrenal tumor as a possible explanation for cortisol hypersecretion and tumor growth in Cushing s syndrome during pregnancy. A 27-yr-old woman presented with hypertension and diabetes mellitus in early pregnancy. Investigations revealed hypercortisolemia, suppressed ACTH-levels, and a 6.4- cm right adrenal tumor. The tumor was successfully removed by laparoscopy at 26th week of pregnancy. Hypercortisolism and hypertension resolved post-operatively. The tumor displayed higher LH/hCG receptor mRNA and protein positivity than adjacent normal adrenal tissue as examined by in situ hybridization and immunocytochemistry. High physiological levels of hCG, in conjunction with aberrant adrenal LH/hCG receptor overexpression, may have contributed to the development of Cushing's syndrome in pregnancy.

Increased ratio of mRNA expression of the genes CYP17 and CYP11B1 indicates autonomous cortisol production in adrenocortical tumors.

OBJECTIVE: Due to increased use of imaging techniques, adrenal incidentalomas are frequently detected. The majority are non-hyperfunctioning adrenocortical tumors. We have previously shown that expression of the gene CYP17, coding for the enzyme in the cortisol pathway, correlates with cortisol release from adrenocortical tumors in vitro. The aim of this study was to compare clinical data with mRNA expression of CYP17 and CYP11B1 in adrenocortical tumors from patients with and without Cushing's syndrome and to identify adrenal tumors that may cause subclinical Cushing's syndrome. DESIGN: A retrospective study of 34 patients undergoing adrenalectomy due to an adrenal tumor. METHODS: Clinical data were collected. In the adrenal gland the mRNA expression of the genes CYP17 and CYP11B1 was studied with in situ hybridisation technique. RESULTS: The median ratio of CYP17/CYP11B1 expression in tumors from patients with Cushing's syndrome was significantly higher than the median ratio in the non-hyperfunctioning tumors. Tumors from 2 patients with subclinical Cushing's syndrome had ratios within the upper range for non-hyperfunctioning tumors. CONCLUSIONS: The ratio between the expression of the genes CYP17 and CYP11B1 in tumors from patients with Cushing's syndrome is significantly higher than in the non-hyperfunctioning tumors. This indicates that 17alpha-hydroxylase is a major determinant of cortisol overproduction. The patients with subclinical Cushing's syndrome in this study are too few to draw any firm conclusions although the results suggest that subclinical Cushing's syndrome may be identified post-operatively with this method.

Mechanisms of hypotonic inhibition of the sodium, proton exchanger type 1 (NHE1) in a biliary epithelial cell line (Mz-Cha-1).

AIM: To elucidate the cellular events that results in inhibition of Na(+), H(+) exchanger type 1 (NHE1) by hypotonicity. METHODS: Intracellular pH (pH(i)) was measured in biliary epithelial cells, with the pH-sensitive fluorochrome 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) using a spectrophotometer. Regulatory volume decrease (RVD) was analysed from confocal images. Changes in NHE1 membrane content were visualized by confocal laser scanning microscopy after transfection of Mz-Cha-1 cells with a NHE1-cMyc fusion protein. RESULTS: In Mz-Cha-1 cells hypotonicity (-80 mmol L(-1) NaCl) inhibited endogenous Na(+), H(+) exchange. Tyrosine and serine kinase inhibitors were incapable to prevent inhibition. As several signalling pathways influence Na(+), H(+) exchange, we tested the effect of the Ca(++), Calmodulin, protein kinase C or the cAMP, protein kinase A system on inhibition of Na(+), H(+) exchange by hypotonic challenge, but neither system was involved. In contrast, cytoskeleton did influence the effect of hypotonicity. Inhibition of microtubule polymerization by colchicine prevented inhibition of NHE1, and also restored Na(+), H(+) exchange kinetics. Specific inhibition of Src kinases with PP2, attenuated pH(i) recovery rate from 1.93 +/- 0.16 pH units min(-1) (normotonic environment) to 1.02 +/- 0.50 pH units min(-1) (hypotonic environment). Membrane staining of NHE1-cMyc fusion protein was maintained after hypotonic exposure in colchicine pre-treated cells as was RVD. Microfilament inhibition by cytochalasin preserved NHE1 activity. Inhibition of phosphatidylinositol-3'-kinase was unable to restore Na(+), H(+) exchange activity. CONCLUSION: We conclude that regulation of Na(+), H(+) exchange during RVD is mediated by cytoskeletal elements. This receptor independent pathway is regulated by Src.

Endoscopic ultrasonography for evaluation of pancreatic tumours in multiple endocrine neoplasia type 1.

BACKGROUND: Pancreatic tumours are common in patients with multiple endocrine neoplasia type 1 (MEN1), and close surveillance is needed to detect pancreatic lesions at an early stage. Conventional radiology is inefficient in verifying the small tumours indicated by biochemical screening. During the past decade, endoscopic ultrasonography (EUS) has evolved as a sensitive method for the detection of small pancreatic lesions. METHODS: EUS was evaluated in 25 patients with MEN1, two of whom had symptoms due to hormonal secretion. Twenty-two patients had biochemical signs of pancreatic tumours, and in five patients lesions were located by either computed tomography (two) or transabdominal ultrasonography (three). RESULTS: EUS visualized pancreatic tumours in the five patients in whom lesions were detected by the other methods and in a further nine patients. Eight of these 14 patients had surgery, and tumours were confirmed histopathologically. No lesion was detected in any of the 11 patients with no tumour detected by EUS. CONCLUSION: EUS is a more sensitive technique for the detection and localization of potentially malignant lesions in patients with MEN1 than computed tomography or transabdominal ultrasonography.

A 10-kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b.

The major structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) are derived from ORFs 5, 6, and 7. Western blots of sucrose gradient-purified virions and PRRSV-infected MARC-145 cells, probed with immune pig serum, showed the presence of an additional 10-kDa protein. Nucleotide sequence analysis of North American PRRSV isolate SDSU-23983 revealed a small ORF within ORF2, named ORF2b, which, when translated, produced a 73-amino-acid nonglycosylated protein. Recombinant 2b protein expressed by a baculovirus clone, AcVR2, comigrated with the 10-kDa virus-associated protein. The loss of 10-kDa protein immunoreactivity after absorption of immune sera with lysates from AcVR2-infected insect cells demonstrated that the 2b and 10-kDa proteins are immunologically similar. Immunoblots were also used for the detection of anti-2b activity in serum samples from experimentally infected adult pigs. Antibodies against PRRSV were apparent by 14 days postinfection, followed by anti-2b activity and serum neutralizing activity. The putative ORF2b start codon is only 6 nucleotides downstream of the adenine of the ORF2a start codon. The expression of ORF2a and 2b as enhanced green fluorescent fusion proteins showed that both proteins were translated; however, the ORF2b was preferentially expressed. These results suggest that the 2b protein is virion associated and the principal product of ORF2.

Identification of porcine reproductive and respiratory syndrome virus in semen and tissues from vasectomized and nonvasectomized boars.

Previous studies have indicated that porcine reproductive and respiratory syndrome virus (PRRSV) can be identified in and transmitted through boar semen. However, the site(s) of replication indicating the origin of PRRSV in semen has not been identified. To determine how PRRSV enters boar semen, five vasectomized and two nonvasectomized PRRSV-seronegative boars were intranasally inoculated with PRRSV isolate VR-2332. Semen was collected three times weekly from each boar and separated into cellular and cell-free (seminal plasma) fractions. Both fractions were evaluated by reverse transcriptase nested polymerase chain reaction (RT-nPCR) for the presence of PRRSV RNA. Viremia and serostatus were evaluated once weekly, and boars were euthanatized 21 days postinoculation (DPI). Tissues were collected and evaluated by RT-nPCR, virus isolation (VI), and immunohistochemistry to identify PRRSV RNA, infectious virus, or viral antigen, respectively. PRRSV RNA was identified in semen from all vasectomized and nonvasectomized boars and was most consistently found in the cell fraction, within cells identified with a macrophage marker. Viral replication as determined by VI was predominately found within lymphoid tissue. However, PRRSV RNA was widely disseminated throughout many tissues, including the reproductive tract at 21 DPI. These results indicate that PRRSV can enter semen independent of testicular or epididymal tissues, and the source of PRRSV in semen is virus-infected monocytes/macrophages or non-cell-associated virus in serum. PRRSV-infected macrophages in semen may result from infection of local tissue macrophages or may originate from PRRSV-infected circulating monocytes or macrophages.

Pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs.

The pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV) was determined in gnotobiotic pigs by studying the sequential development of microscopic lesions and sites of virus distribution and replication. Thirty-two pigs (three pigs/infected group and one pig/control group) were inoculated by nasal instillation of either PRRSV isolate ATCC VR-2332 (total dose 10(2.6) TCID50) or uninfected cell culture supernatant. Infected and control pigs were euthanized at 12 hours, and 1, 2, 3, 5, 7, 14, and 21 days postexposure (PE). Gnotobiotic pigs experimentally infected with PRRSV were viremic by 12 hours PE and subsequently developed pneumonia, lymphadenopathy, vasculitis, myocarditis and encephalitis. Lung lesions developed by day 3 PE, persisted through day 21 PE and were characterized by alveolar septa thickened by macrophages, alveolar proteinaceous and karyorrhectic debris, alveolar syncytial cells, and multifocal type II pneumocyte hypertrophy. Lymph node lesions varied in distribution and severity and were characterized by germinal center hypertrophy and hyperplasia, lymphocyte necrosis, multiple cystic spaces, and polykaryocytes within the cystic spaces. Heart lesions were a late feature of infection and all infected pigs had heart lesions on day 21 PE characterized by subendocardial, myocardial, and perivascular foci of lymphocytes. Vasculitis also varied in distribution and severity and affected all sizes of vessels. Results of this experiment indicate that PRRSV is a multisystem disease characterized initially by viremia with subsequent virus distribution and replication in multiple organs causing interstitial pneumonia, vasculitis, lymphadenopathy, myocarditis, and encephalitis.