Submandibular glands and the regulation of gastric proliferation and TGFα distribution during rat postnatal development

Rodent gastric mucosa grows and differentiates during suckling-weaning transition. Among the molecules in rat milk, EGF and TGFβ are important peptides in the control of cell proliferation, and together with TGFα, they are also produced by submandibular glands. We aimed to determine the effect of saliva and milk on epithelial cell proliferation in the stomach of rat pups. We also examined the distribution of TGFα in the gastric mucosa after sialoadenectomy (SIALO) and fasting in order to determine whether this growth factor is affected by the deprivation of molecules derived from saliva and milk. SIALO was performed at 14 days and fasting was induced 3 days later. Cell proliferation was evaluated through metaphasic index and TGFα was detected by immunohistochemistry. We observed that whereas SIALO did not alter cell division, since the metaphasic index (MI) was unchanged, fasting stimulated cell proliferation (P < 0.05). After SIALO and fasting, MI was reduced when compared to the fasted group (P < 0.05). We found that TGFα is distributed along gastric gland and SIALO did not interfere in the localization and number of immunolabeled cells, but fasting increased their density when compared to the control (P < 0.05). The association of SIALO and fasting reduced TGFα immunostaining (P < 0.05). Therefore, during fasting, high MI was parallel to increased TGFα in gastric epithelium, but interestingly, this effect was found only in the presence of submandibular glands. We suggest that during suckling, peptides derived from saliva and milk are important to regulate gastric growth.

Nutrient Transport in the Mammary Gland: Calcium, Trace Minerals and Water Soluble Vitamins.

Milk nutrients are secreted by epithelial cells in the alveoli of the mammary gland by several complex and highly coordinated systems. Many of these nutrients are transported from the blood to the milk via transcellular pathways that involve the concerted activity of transport proteins on the apical and basolateral membranes of mammary epithelial cells. In this review, we focus on transport mechanisms that contribute to the secretion of calcium, trace minerals and water soluble vitamins into milk with particular focus on the role of transporters of the SLC series as well as calcium transport proteins (ion channels and pumps). Numerous members of the SLC family are involved in the regulation of essential nutrients in the milk, such as the divalent metal transporter-1 (SLC11A2), ferroportin-1 (SLC40A1) and the copper transporter CTR1 (SLC31A1). A deeper understanding of the physiology and pathophysiology of these transporters will be of great value for drug discovery and treatment of breast diseases.

Identification and evaluation of candidate genes for inherited retinal -degenerative diseases

Although more than 100 genes associated with inherited retinal disease have been mapped to chromosomal locations, less than half of these genes have been cloned. This text includes identification and evaluation of candidate genes for three autosomal dominant forms of inherited retinal degeneration: atypical vitelliform macular dystrophy (VMD1), cone-rod dystrophy (CORD), and retinitis pigmentosa (RP). ^ VMD1 is a disorder characterized by complete penetrance but extremely variable expressivity, and includes macular or peripheral retinal lesions and peripappilary abnormalitites. In 1984, linkage was reported between VMD1 and soluble glutamate-pyruvate transaminase GPT); however, placement of GPT to 8q24 on linkage maps had been debated, and VMD1 did not show linkage to microsatellite markers in that region. This study excluded linkage between the loci by cloning GPT, identifying the nucleotide substitution associated with the GPT sozymes, and by assaying VMD1 family samples with an RFLP designed to detect the substitution. In addition, linkage of VMD1 to the known dominant macular degeneration loci was excluded. ^ CORD is characterized by early onset of color-vision deficiency, and decreased visual acuity, However, this retinal degeneration progresses to no light perception, severe macular lesion, and “bone-spicule” accumulations in the peripheral retina. In this study, the disorder in a large Texan family was mapped to the CORD2 locus of 19q13, and a mutation in the retina/pineal-specific cone-rod homeobox gene (CRX) was identified as the disease cause. In addition, mutations in CRX were associated with significantly different retinal disease phenotypes, including retinitis pigmentosa and Leber congenital amaurosis. ^ Many of the mutations leading to inherited retinal disorders have been identified in genes like CRX, which are expressed predominantly in the retina and pineal gland. Therefore, a combination of database analysis and laboratory investigation was used to identify 26 novel retina/pineal-specific expressed sequence tag (EST) clusters as candidate genes for inherited retinal disorders. Eight of these genes were mapped into the candidate regions of inherited retinal degeneration loci. ^ Two of the eight clusters mapped into the retinitis pigmentosa RP13 candidate region of 17p13, and were both determined to represent a single gene that is highly expressed in photoreceptors. This gene, the Ah receptor-interacting like protein-1 (AIPL1), was cloned, characterized, and screened for mutations in RP13 patient DNA samples. ^

The organs of internal secretion, their diseases and therapeutic application; a book for general practitioners.

"In this edition several important alterations have been made in the construction of the book. A chapter has been inserted dealing with the physiology of the internal secretions and their relationships ... The position of this chapter has necessitated the renumbering of the subsequent chapters; and two chapters have been added in later parts of the book." - Pref.

Histological analysis of gallbladder diseases in relation to opisthorchiasis in endemic areas of Thailand

Chronic gallbladder disease frequently accompanies infection with the liver fluke, Opisthorchis viverrini, in Northeast Thailand. However, the pathology and pathogenesis of the gallbladder disease have not been described. Accordingly, gallbladder specimens from 187 consecutive patients who had undergone cholecystectomy at a referral hospital in an endemic area in Thailand were histologically characterized in relation to O. viverrini infection. The infection was assessed by the presence of parasite eggs in the bile and/or antibody response to the liver fluke. The average level of parasite-specific IgG was significantly higher in patients with Opisthorchis eggs in the bile than those without (P < 0.001). The main histopathologic features of the gallbladder included inflammation, mucosal atrophy/or hyperplasia, goblet cell metaplasia, mucous gland hyperplasia, Rokitansky-Aschoff sinus formation, dysplasia and fibrosis. The fibrosis was strongly associated with elevated levels of Opisthorchis-specific antibody (P < 0.001) but not with the presence of parasite eggs. Other pathologic features did not vary in frequency or severity with parasitological status. Our results show that severe fibrosis of the gallbladder is a more common histologic feature of cholecystitis among those with O. viverrini infection compared to those without infection. The close relationship between parasite-specific IgG and severe fibrosis suggests that specific immune response to the parasite play an important role in the pathogenesis of the fibrotic change. (C) 2003 Elsevier B.V. All rights reserved.

Brca1 inactivation induces p27Kip1-dependent cell cycle arrest and delayed development in the mouse mammary gland

One common characteristic of breast cancers arising in carriers of the predisposition gene BRCA1 is a loss of expression of the CDK inhibitor p27(Kip1) (p27), suggesting that p27 interacts epistatically with BRCA1. To investigate this relationship, we examined expression of p27 in mice expressing a dominant negative allele of Brca1 (MMTV-trBr) in the mammary gland. While these mice rarely develop tumors, they showed a 50% increase in p27 protein and a delay in mammary gland development associated with reduced proliferation. In contrast, on a p27 heterozygote background, MMTV-trBrca1 mice showed an increase in S phase cells, and normal mammary development. p27 was the only protein in the cyclin cyclin-dependent kinase network to show altered expression, suggesting that it may be a central mediator of cell cycle arrest in response to loss of function of BRCA1. Furthermore, in human mammary epithelial MCF7 cells expressing BRCA1-specific RNAi and in the BRCA1-deficient human tumor cell line HCC1937, p27 is elevated at the mRNA level compared to cells expressing wild-type BRCA1. We hypothesize that disruption of BRCA1 induces an increase in p27 that inhibits proliferation. Accordingly, reduction in p27 expression leads to enhancement of cellular proliferation in the absence of BRCA1.

Prolactin and the expression of suppressor of cytokine signaling-3 in the sheep adrenal gland before birth

Prolactin and the expression of suppressor of cytokine signaling-3 in the sheep adrenal gland before birth. Am J Physiol Regul Integr Comp Physiol 291: R1399-R1405, 2006. First published June 29, 2006; doi: 10.1152/ajpregu.00252.2006.-The fetal pituitary-adrenal axis plays a key role in the fetal response to intrauterine stress and in the timing of parturition. The fetal sheep adrenal gland is relatively refractory to stimulation in midgestation (90-120 days) before the prepartum activation, which occurs around 135 days gestation (term = 147 +/- 3 days). The mechanisms underlying the switch from adrenal quiescence to activation are unclear. Therefore, we have investigated the expression of suppressor of cytokine signaling-3 (SOCS-3), a putative inhibitor of tissue growth in the fetal sheep adrenal between 50 and 145 days gestation and in the adrenal of the growth-restricted fetal sheep in late gestation. SOCS-3 is activated by a range of cytokines, including prolactin (PRL), and we have, therefore, determined whether PRL administered in vivo or in vitro stimulates SOCS-3 mRNA expression in the fetal adrenal in late gestation. There was a decrease (P < 0.005) in SOCS-3 expression in the fetal adrenal between 54 and 133 days and between 141 and 144 days gestation. Infusion of the dopaminergic agonist, bromocriptine, which suppressed fetal PRL concentrations but did not decrease adrenal SOCS-3 mRNA expression. PRL administration, however, significantly increased adrenal SOCS-3 mRNA expression (P < 0.05). Similarly, there was an increase (P < 0.05) in SOCS-3 mRNA expression in adrenocortical cells in vitro after exposure to PRL (50 ng/ml). Placental and fetal growth restriction had no effect on SOCS-3 expression in the adrenal during late gestation. In summary, the decrease in the expression of the inhibitor SOCS-3 after 133 days gestation may be permissive for a subsequent increase in fetal adrenal growth before birth. We conclude that factors other than PRL act to maintain adrenal SOCS-3 mRNA expression before 133 days gestation but that acute elevations of PRL can act to upregulate adrenal SOCS-3 expression in the sheep fetus during late gestation.

MicroRNA modulation of aldosterone production in the adrenal gland

Hypertension is the major risk factor for coronary disease worldwide. Primary hypertension is idiopathic in origin but is thought to arise from multiple risk factors including genetic, lifestyle and environmental influences. Secondary hypertension has a more definite aetiology; its major single cause is primary aldosteronism (PA), the greatest proportion of which is caused by aldosteroneproducing adenoma (APA), where aldosterone is synthesized at high levels by an adenoma of the adrenal gland. There is strong evidence to show that high aldosterone levels cause adverse effects on cardiovascular, cerebrovascular, renal and other systems. Extensive studies have been conducted to analyse the role that regulation of CYP11B2, the gene encoding the aldosterone synthase enzyme plays in determining aldosterone production and the development of hypertension. One significant regulatory factor that has only recently emerged is microRNA (miRNA). miRNAs are small non-coding RNAs, synthesized by a series of enzymatic processes, that negatively regulate gene expression at the posttranscriptional level. Detection and manipulation of miRNA is now known to be a viable method in the treatment, prevention and prognosis of certain diseases. The aim of the present study was to identify miRNAs likely to have a role in the regulation of corticosteroid biosynthesis. To achieve this, the miRNA profile of APA and normal human adrenal tissue was compared, as was the H295R adrenocortical cell line model of adrenocortical function, under both basal conditions and following stimulation of aldosterone production. Key differentially-expressed miRNAs were then identified and bioinformatic tools used to identify likely mRNA targets and pathways for these miRNAs, several of which were investigated and validated using in vitro methods. The background to this study is set out in Chapter 1 of this thesis, followed by a description of the major technical methods employed in Chapter 2. Chapter 3 presents the first of the study results, analysing differences in miRNA profile between APA and normal human adrenal tissue. Microarray was implemented to detect the expression of miRNAs in these two tissue types and several miRNAs were found to vary significantly and consistently between them. Furthermore, members of several miRNA clusters exhibited similar changes in expression pattern between the two tissues e.g. members of cluster miR-29b-1 (miR-29a-3p and miR-29b-3p) and of cluster miR-29b-2 (miR-29b-3p and miR-29c- 3p) are downregulated in APA, while members of cluster let-7a-1 (let-7a-5p and let-7d-5p), cluster let-7a-3 (let-7a-5p and let-7b-5p) and cluster miR-134 (miR- 134 and miR-382) are upregulated. Further bioinformatic analysis explored the possible biological function of these miRNAs using Ingenuity® Systems Pathway Analysis software. This led to the identification of validated mRNAs already known to be targeted by these miRNAs, as well as the prediction of other mRNAs that are likely targets and which are involved in processes relevant to APA pathology including cholesterol synthesis (HMGCR) and corticosteroidogenesis (CYP11B2). It was therefore hypothesised that increases in miR-125a-5p or miR- 335-5p would reduce HMGCR and CYP11B2 expression. Chapter 4 describes the characterisation of H295R cells of different strains and sources (H295R Strain 1, 2, 3 and HAC 15). Expression of CYP11B2 was assessed following application of 3 different stimulants: Angio II, dbcAMP and KCl. The most responsive strain to stimulation was Strain 1 at lower passage numbers. Furthermore, H295R proliferation increased following Angio II stimulation. In Chapter 5, the hypothesis that increases in miR-125a-5p or miR-335-5p reduces HMGCR and CYP11B2 expression was tested using realtime quantitative RT-PCR and transfection of miRNA mimics and inhibitors into the H295R cell line model of adrenocortical function. In this way, miR-125a-5p and miR-335-5p were shown to downregulate CYP11B2 and HMGCR expression, thereby validating certain of the bioinformatic predictions generated in Chapter 3. The study of miRNA profile in the H295R cell lines was conducted in Chapter 6, analysing how it changes under conditions that increase aldosterone secretion, including stimulation Angiotensin II, potassium chloride or dibutyryl cAMP (as a substitute for adrenocorticotropic hormone). miRNA profiling identified 7 miRNAs that are consistently downregulated by all three stimuli relative to basal cells: miR-106a-5p, miR-154-3p, miR-17-5p, miR-196b-5p, miR-19a-3p, miR-20b- 5p and miR-766-3p. These miRNAs include those derived from cluster miR-106a- 5p/miR-20b-5p and cluster miR-17-5p/miR-19a-3p, each producing a single polycistronic transcript. IPA bioinformatic analysis was again applied to identify experimentally validated and predicted mRNA targets of these miRNAs and the key biological pathways likely to be affected. This predicted several interactions between miRNAs derived from cluster miR-17-5p/miR-19a-3p and important mRNAs involved in cholesterol biosynthesis: LDLR and ABCA1. These predictions were investigated by in vitro experiment. miR-17-5p/miR-106a-p and miR-20b-5p were found to be consistently downregulated by stimulation of aldosterone biosynthesis. Moreover, miR-766-3p was upregulation throughout. Furthermore, I was able to validate the downregulation of LDLR by miR-17 transfection, as predicted by IPA. In summary, this study identified key miRNAs that are differentially-expressed in vivo in cases of APA or in vitro following stimulation of aldosterone biosynthesis. The many possible biological actions these miRNAs could have were filtered by bioinformatic analysis and selected interactions validated in vitro. While direct actions of these miRNAs on steroidogenic enzymes were identified, cholesterol handling also emerged as an important target and may represent a useful point of intervention in future therapies designed to modulate aldosterone biosynthesis and reduce its harmful effects.

High Diagnostic Accuracy And Reproducibility Of Fine-needle Aspiration Cytology For Diagnosing Salivary Gland Tumors: Cytohistologic Correlation In 182 Cases.

The purpose of this study was to assess the efficacy and reproducibility of the cytologic diagnosis of salivary gland tumors (SGTs) using fine-needle aspiration cytology (FNAC). The study aimed to determine diagnostic accuracy, sensitivity, and specificity and to evaluate the extent of interobserver agreement. We retrospectively evaluated SGTs from the files of the Division of Pathology at the Clinics Hospital of São Paulo and Piracicaba Dental School between 2000 and 2006. We performed cytohistologic correlation in 182 SGTs. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were 94%, 100%, 100%, 100%, and 99%, respectively. The interobserver cytologic reproducibility showed significant statistical concordance (P < .0001). FNAC is an effective tool for performing a reliable preoperative diagnosis in SGTs and shows high diagnostic accuracy and consistent interobserver reproducibility. Further FNAC studies analyzing large samples of malignant SGTs and reactive salivary lesions are needed to confirm their accuracy.

Otorhinolaryngological Findings In A Group Of Patients With Rheumatic Diseases.

Otorhinolaryngological manifestations of rheumatologic diseases represent a great challenge not only to the generalistphysician but also to the ENT doctor andrheumatologist. They often represent early manifestations of an autoimmune disorder which requires prompt and aggressive immunosuppressive treatment. Auditory, nasal, laryngeal and eye symptoms can be the first manifestation of rheumatic diseases and their proper assessment helps the doctor to identify signs of disease activity. The objective of this study is to identify the ENT manifestations in patients with rheumatic diseases in a high complexity hospital, regarding facilitating an early diagnosis and treatment. We performed clinical and complete otorhinolaryngological evaluations in patients selected from the outpatient rheumatology in a standardized manner by the use of a standardized form filling during the secondhalf of 2010. In the study group, systemic lupus erythematosus (SLE) patients had predominantly laryngeal manifestations, while patients with Sjögren's syndrome showed a higher prevalence of otologic manifestations. Changes in audiometric tests were found in 53% of Wegener's granulomatosis (WG) patients, 80% of relapsing polychondritis (RP), 33% of systemic lupus erythematosus (SLE) and 50% of Churg-Strauss syndrome (SCS). Regarding nasal alterations, these were found so prevalent in all conditions, especially Churg-Strauss syndrome. This study demonstrated that most patients treated in our hospital has the ENT signs and symptoms commonly associated in previous studies on rheumatic diseases, but further studies with a larger number of patients must be made to establish such relations.

Volumetric Capnography For The Evaluation Of Chronic Airways Diseases.

Obstructive lung diseases of different etiologies present with progressive peripheral airway involvement. The peripheral airways, known as the silent lung zone, are not adequately evaluated with conventional function tests. The principle of gas washout has been used to detect pulmonary ventilation inhomogeneity and to estimate the location of the underlying disease process. Volumetric capnography (VC) analyzes the pattern of CO2 elimination as a function of expired volume. To measure normalized phase 3 slopes with VC in patients with non-cystic fibrosis bronchiectasis (NCB) and in bronchitic patients with chronic obstructive pulmonary disease (COPD) in order to compare the slopes obtained for the groups. NCB and severe COPD were enrolled sequentially from an outpatient clinic (Hospital of the State University of Campinas). A control group was established for the NCB group, paired by sex and age. All subjects performed spirometry, VC, and the 6-Minute Walk Test (6MWT). Two comparisons were made: NCB group versus its control group, and NCB group versus COPD group. The project was approved by the ethical committee of the institution. Statistical tests used were Wilcoxon or Student's t-test; P<0.05 was considered to be a statistically significant difference. Concerning the NCB group (N=20) versus the control group (N=20), significant differences were found in body mass index and in several functional variables (spirometric, VC, 6MWT) with worse results observed in the NCB group. In the comparison between the COPD group (N=20) versus the NCB group, although patients with COPD had worse spirometric and 6MWT values, the capnographic variables mean phase 2 slope (Slp2), mean phase 3 slope normalized by the mean expiratory volume, or mean phase 3 slope normalized by the end-tidal CO2 concentration were similar. These findings may indicate that the gas elimination curves are not sensitive enough to monitor the severity of structural abnormalities. The role of normalized phase 3 slope may be worth exploring as a more sensitive index of small airway disease, even though it may not be equally sensitive in discriminating the severity of the alterations.

Systemic Granulomatous Diseases Associated With Multiple Palpable Masses That May Involve The Breast: Case Presentation And An Approach To The Differential Diagnosis.

Palpable mass is a common complaint presented to the breast surgeon. It is very uncommon for patients to report breast mass associated with palpable masses in other superficial structures. When these masses are related to systemic granulomatous diseases, the diagnosis and initiation of specific therapy can be challenging. The purpose of this paper is to report a case initially assessed by the breast surgeon and ultimately diagnosed as granulomatous variant of T-cell lymphoma, and discuss the main systemic granulomatous diseases associated with palpable masses involving the breast.

Tubular Variant Of Basal Cell Adenoma Shares Immunophenotypical Features With Normal Intercalated Ducts And Is Closely Related To Intercalated Duct Lesions Of Salivary Gland.

The morphological criteria for identification of intercalated duct lesions (IDLs) of salivary glands have been defined recently. It has been hypothesised that IDL could be a precursor of basal cell adenoma (BCA). BCAs show a variety of histological patterns, and the tubular variant is the one that presents the strongest resemblance with IDLs. The aim of this study was to analyse the morphological and immunohistochemical profiles of IDLs and BCAs classified into tubular and non-tubular subtypes, to determine whether or not IDL and tubular BCA represent distinct entities. Eight IDLs, nine tubular BCAs and 19 non-tubular BCAs were studied. All tubular BCAs contained IDL-like areas, which represented 20-70% of the tumour. In non-tubular BCA, IDL-like areas were occasional and small (<5%). One patient presented IDLs, tubular BCAs and IDL/tubular BCA combined lesions. Luminal ductal cells of IDLs and tubular BCAs exhibited positivity for CK7, lysozyme, S100 and DOG1. In the non-tubular BCA group, few luminal cells exhibited such an immunoprofile; they were mainly CK14-positive. Basal/myoepithelial cells of IDLs, tubular BCAs and non-tubular BCAs were positive for CK14, calponin, α-SMA and p63; they were more numerous in BCA lesions. IDL, tubular BCA and non-tubular BCA form a continuum of lesions in which IDLs are related closely to tubular BCA. In both, the immunoprofile of luminal and myoepithelial cells recapitulates the normal intercalated duct. The difference between the adenoma-like subset of IDLs and tubular BCA rests mainly on the larger numbers of myoepithelial cells in the latter. Our findings indicate that at least some BCAs can arise via IDLs.

Cheaper Faster Drug Development Validated By The Repositioning Of Drugs Against Neglected Tropical Diseases.

There is an urgent need to make drug discovery cheaper and faster. This will enable the development of treatments for diseases currently neglected for economic reasons, such as tropical and orphan diseases, and generally increase the supply of new drugs. Here, we report the Robot Scientist 'Eve' designed to make drug discovery more economical. A Robot Scientist is a laboratory automation system that uses artificial intelligence (AI) techniques to discover scientific knowledge through cycles of experimentation. Eve integrates and automates library-screening, hit-confirmation, and lead generation through cycles of quantitative structure activity relationship learning and testing. Using econometric modelling we demonstrate that the use of AI to select compounds economically outperforms standard drug screening. For further efficiency Eve uses a standardized form of assay to compute Boolean functions of compound properties. These assays can be quickly and cheaply engineered using synthetic biology, enabling more targets to be assayed for a given budget. Eve has repositioned several drugs against specific targets in parasites that cause tropical diseases. One validated discovery is that the anti-cancer compound TNP-470 is a potent inhibitor of dihydrofolate reductase from the malaria-causing parasite Plasmodium vivax.