Immunohistochemical expression of nuclear factor B, matrix metalloproteinase 9, and endoglin (CD105) in odontogenic keratocysts, dentigerous cysts, and radicular cysts

OBJECTIVE: The aim of this study was to compare the immunohistochemical expression of nuclear factor κB (NF-κB), matrix metalloproteinase 9 (MMP-9), and CD105 in odontogenic keratocysts (OKCs), dentigerous cysts (DCs), and radicular cysts (RCs). STUDY DESIGN: Twenty cases of OKCs, 20 DCs, and 20 RCs were analyzed. A labeling index (LI), which expresses the percentage of NF-κB-stained nuclei, was calculated for the analysis of NF-κB expression. Expression of MMP-9 in the epithelium and in the capsule of each lesion was scored as 0 (<10% stained cells), 1 (10%-50% stained cells), or 2 (>50% stained cells). In addition, MMP-9 immunostaining was analyzed in endothelial cells of vessels with a conspicuous lumen. The angiogenic index was determined based on the number of anti-CD105 antibody-stained microvessels. RESULTS: In the epithelial component, the NF-κB LI was higher in OKCs than in DCs and RCs (P < .001). Analysis of MMP-9 expression in the epithelial component showed a predominance of score 2 in OKCs (90%), DCs (70%), and RCs (65%; P = .159). Evaluation of the NF-κB LI according to the expression of MMP-9 in the epithelial lining revealed no significant difference between lesions (P = .282). In the fibrous capsule, the highest percentage of MMP-9-stained cells (score 2) was observed in OKCs (P = .100). Analysis of the expression of MMP-9 in the vessels of odontogenic cysts showed a predominance of score 2 in OKCs (80%) and RCs (50%) and of score 1 in DCs (75%; P = .002). Mean microvessel count was high in RCs (16.9), followed by DCs (12.1) and OKCs (10.0; P = .163). No significant difference in microvessel count according to the expression of MMP-9 was observed between groups (P = .689). CONCLUSIONS: The results suggest that the more aggressive biologic behavior of OKCs is related to the higher expression of MMP-9 and NF-κB in those lesions. The differences in the biologic behavior of the lesions studied do not seem to be associated with the angiogenic index.

Dynamic Compartmentalization of Persistent UPEC in the Superficial Bladder Epithelium

Urinary tract infections (UTIs) are typically caused by bacteria that colonize different regions of the urinary tract, mainly the bladder and the kidney. Approximately 25% of women that suffer from UTIs experience a recurrent infection within 6 months of the initial bout, making UTIs a serious economic burden resulting in more than 10 million hospital visits and $3.5 billion in healthcare costs in the United States alone. Type-1 fimbriated Uropathogenic E. coli (UPEC) is the major causative agent of UTIs, accounting for almost 90 % of bacterial UTIs. The unique ability of UPEC to bind and invade the superficial bladder epithelium allows the bacteria to persist inside epithelial niches and survive antibiotic treatment. Persistent, intracellular UPEC are retained in the bladder epithelium for long periods, making them a source of recurrent UTIs. Hence, the ability of UPEC to persist in the bladder is a matter of major health and economic concern, making studies exploring the underlying mechanism of UPEC persistence highly relevant.

In my thesis, I will describe how intracellular Uropathogenic E.coli (UPEC) evade host defense mechanisms in the superficial bladder epithelium. I will also describe some of the unique traits of persistent UPEC and explore strategies to induce their clearance from the bladder. I have discovered that the UPEC virulence factor Alpha-hemolysin (HlyA) plays a key role in the survival and persistence of UPEC in the superficial bladder epithelium. In-vitro and in-vivo studies comparing intracellular survival of wild type (WT) and hemolysin deficient UPEC suggested that HlyA is vital for UPEC persistence in the superficial bladder epithelium. Further in-vitro studies revealed that hemolysin helped UPEC persist intracellularly by evading the bacterial expulsion actions of the bladder cells and remarkably, this virulence factor also helped bacteria avoid t degradation in lysosomes.

To elucidate the mechanistic basis for how hemolysin promotes UPEC persistence in the urothelium, we initially focused on how hemolysin facilitates the evasion of UPEC expulsion from bladder cells. We found that upon entry, UPEC were encased in “exocytic vesicles” but as a result of HlyA expression these bacteria escaped these vesicles and entered the cytosol. Consequently, these bacteria were able to avoid expulsion by the cellular export machinery.

Since bacteria found in the cytosol of host cells are typically recognized by the cellular autophagy pathway and transported to the lysosomes where they are degraded, we explored why this was not the case here. We observed that although cytosolic HlyA expressing UPEC were recognized and encased by the autophagy system and transported to lysosomes, the bacteria appeared to avoid degradation in these normally degradative compartments. A closer examination of the bacteria containing lysosomes revealed that they lacked V-ATPase. V-ATPase is a well-known proton pump essential for the acidification of mammalian intracellular degradative compartments, allowing for the proper functioning of degradative proteases. The absence of V-ATPase appeared to be due to hemolysin mediated alteration of the bladder cell F-actin network. From these studies, it is clear that UPEC hemolysin facilitates UPEC persistence in the superficial bladder epithelium by helping bacteria avoid expulsion by the exocytic machinery of the cell and at the same time enabling the bacteria avoid degradation when the bacteria are shuttled into the lysosomes.

Interestingly even though UPEC appear to avoid elimination from the bladder cell their ability to multiple in bladder cells seem limited.. Indeed, our in-vitro and in-vivo experiments reveal that UPEC survive in superficial bladder epithelium for extended periods of time without a significantly change in CFU numbers. Indeed, we observed these bacteria appeared quiescent in nature. This observation was supported by the observation that UPEC genetically unable to enter a quiescence phase exhibited limited ability to persist in bladder cells in vitro and in vivo, in the mouse bladder.

The studies elucidated in this thesis reveal how UPEC toxin, Alpha-hemolysin plays a significant role in promoting UPEC persistence via the modulation of the vesicular compartmentalization of UPEC at two different stages of the infection in the superficial bladder epithelium. These results highlight the importance of UPEC Alpha-hemolysin as an essential determinant of UPEC persistence in the urinary bladder.

Regenerative Cues of Myeloid Cells on Pancreatic Epithelium

Thesis (Ph.D.)--University of Washington, 2016-07

Sub lethal mercuric chloride toxicity induced stress related alterations in the epithelial lining of foot of the freshwater mussel Lamellidens marginalis (Lamarck)

Sub lethal (0.2 ppm) mercuric chloride induced stress related histopathological alterations in the epithelial linings of foot (podium) of the edible freshwater mussel Lamellidens marginalis (Lamarck) were studied using histochemical techniques up to 60 days of exposure. The histomorphological changes were manifested only slowly and its intensity was somewhat proportional to the duration of exposure. The immediate response of the exposed mussels was the altered mucous secretion. There was a progressive incorporation of sulphated glycoproteins into the secretory contents of the mucous cells especially in the first half of the experiment. Marked histopathological changes including necrosis, appearance of pyknotic nuclei, sloughing of epithelial cells and appearance of non-tissue spaces, etc., started appearing during the later half of the experiment. The fag end of the experiment, which witnessed prominent histomorphological changes, was accompanied by highly decreased mucous secretion. KEYWORDS: heavy metal toxicity, mercuric chloride, Lamellidens marginalis, freshwater mussel, histopathology.

Structural features of the epididymal region of the domestic duck (Anas plathyrynchos)

A região epididimária do pato doméstico era composta pelos ductúlos eferentes, os quais hitotopologicamente foram caracterizados como dúctulos eferentes proximal e distal e, sequencialmente, pelo ducto epididimal. O epitélio dos dúctulos eferentes era pseudo-estratificado, formado por células colunares. O epitélio dos ductos epididimários mostrou-se também pseudo-estratificado, mas não ciliado. de acordo com as análises histomorfométricas, a média da altura epitelial foi significativamente maior nos dútulos esferentes distais, diferindo das baixas médias de altura epitelial observadas nos dúctulos eferentes proximais e ducto epididimal. A média dos diâmetros máximos e mínimos foi significativamente maior nos dúctulos eferentes proximais, comparativamente as médias dos mesmos diâmetros dos outros dúctulos.

Numb/Numbl-Opo antagonism controls retinal epithelium morphogenesis by regulating integrin endocytosis

Polarized trafficking of adhesion receptors plays a pivotal role in controlling cellular behavior during morphogenesis. Particularly, clathrin-dependent endocytosis of integrins has long been acknowledged as essential for cell migration. However, little is known about the contribution of integrin trafficking to epithelial tissue morphogenesis. Here we show how the transmembrane protein Opo, previously described for its essential role during optic cup folding, plays a fundamental role in this process. Through interaction with the PTB domain of the clathrin adaptors Numb and Numbl via an integrin-like NPxF motif, Opo antagonizes Numb/Numbl function and acts as a negative regulator of integrin endocytosis in vivo. Accordingly, numb/numbl gain-of-function experiments in teleost embryos mimic the retinal malformations observed in opo mutants. We propose that developmental regulator Opo enables polarized integrin localization by modulating Numb/Numbl, thus directing the basal constriction that shapes the vertebrate retina epithelium.

Antiangiogenic And Finasteride Therapies: Responses Of The Prostate Microenvironment In Elderly Mice.

The aim of this study was to evaluate the structural and molecular effects of antiangiogenic therapies and finasteride on the ventral prostate of senile mice. 90 male FVB mice were divided into: Young (18 weeks old) and senile (52 weeks old) groups; finasteride group: finasteride (20mg/kg); SU5416 group: SU5416 (6 mg/kg); TNP-470 group: TNP-470 (15 mg/kg,) and SU5416+TNP-470 group: similar to the SU5416 and TNP-470 groups. After 21 days, prostate ventral lobes were collected for morphological, immunohistochemical and Western blotting analyses. The results demonstrated atrophy, occasional proliferative lesions and inflammatory cells in the prostate during senescence, which were interrupted and/or blocked by treatment with antiangiogenic drugs and finasteride. Decreased AR and endostatin reactivities, and an increase for ER-α, ER-β and VEGF, were seen in the senile group. Decreased VEGF and ER-α reactivities and increased ER-β reactivity were verified in the finasteride, SU5416 groups and especially in SU5416+TNP-470 group. The TNP-470 group showed reduced AR and ER-β protein levels. The senescence favored the occurrence of structural and/or molecular alterations suggesting the onset of malignant lesions, due to the imbalance in the signaling between the epithelium and stroma. The SU5416+TNP-470 treatment was more effective in maintaining the structural, hormonal and angiogenic factor balance in the prostate during senescence, highlighting the signaling of antiproliferation via ER-β.

Malignant Transformation Of An Odontogenic Cyst In A Period Of 10 Years.

Primary intraosseous carcinoma of the jaws (PIOSCC) might arise from odontogenic epithelium, more commonly from a previous odontogenic cyst. The aim of this case is to illustrate that the clinician should consider that an apparent benign dentigerous cyst can suffer malignant transformation and that all material removed from a patient must be evaluated histologically. A 44-year-old man presented in a routine periapical X-ray an impacted lower left third molar with radiolucency over its crown. Ten years later, the patient complained of pain in the same region and the tooth was extracted. After one month, the patient still complained of pain and suffered a fracture of the mandible. A biopsy was performed and carcinoma was diagnosed. The patient was treated surgically with adjuvant radio- and chemotherapy and after 8 years, he is well without signs of recurrences. This report describes a central mandibular carcinoma probably developed from a previous dentigerous cyst.

Defective Apoptosis In Intestinal And Mesenteric Adipose Tissue Of Crohn's Disease Patients.

Crohn's disease (CD) is associated with complex pathogenic pathways involving defects in apoptosis mechanisms. Recently, mesenteric adipose tissue (MAT) has been associated with CD ethiopathology, since adipose thickening is detected close to the affected intestinal area. However, the potential role of altered apoptosis in MAT of CD has not been addressed. To evaluate apoptosis in the intestinal mucosa and MAT of patients with CD. Samples of intestinal mucosa and MAT from patients with ileocecal CD and from non-inflammatory bowel diseases patients (controls) were studied. Apoptosis was assessed by TUNEL assay and correlated with the adipocytes histological morphometric analysis. The transcriptional and protein analysis of selected genes and proteins related to apoptosis were determined. TUNEL assay showed fewer apoptotic cells in CD, when compared to the control groups, both in the intestinal mucosa and in MAT. In addition, the number of apoptotic cells (TUNEL) correlated significantly with the area and perimeter of the adipose cells in MAT. Transcriptomic and proteomic analysis reveal a significantly lower transcript and protein levels of Bax in the intestinal mucosa of CD, compared to the controls; low protein levels of Bax were found localized in the lamina propria and not in the epithelium of this tissue. Furthermore, higher level of Bcl-2 and low level of Caspase 3 were seen in the MAT of CD patients. The defective apoptosis in MAT may explain the singular morphological characteristics of this tissue in CD, which may be implicated in the pathophysiology of the disease.

[the Use Of Fish On Buccal Smear To Investigate Mosaicism With A 45,x Cell Line: Study On Healthy Men And Patients With Disorders Of Sex Development].

To verify whether fluorescence in situ hybridization (FISH) of cells from the buccal epithelium could be employed to detect cryptomosaicism with a 45,X lineage in 46,XY patients. Samples of nineteen 46,XY healthy young men and five patients with disorders of sex development (DSD), four 45,X/46,XY and one 46,XY were used. FISH analysis with X and Y specific probes on interphase nuclei from blood lymphocytes and buccal epithelium were analyzed to investigate the proportion of nuclei containing only the signal of the X chromosome. The frequency of nuclei containing only the X signal in the two tissues of healthy men did not differ (p = 0.69). In all patients with DSD this frequency was significantly higher, and there was no difference between the two tissues (p = 0.38), either. Investigation of mosaicism with a 45,X cell line in patients with 46,XY DSD or sterility can be done by FISH directly using cells from the buccal epithelium.

Endocytosis Of Tight Junctions Caveolin Nitrosylation Dependent Is Improved By Cocoa Via Opioid Receptor On Rpe Cells In Diabetic Conditions.

Retinal pigment epithelium cells, along with tight junction (TJ) proteins, constitute the outer blood retinal barrier (BRB). Contradictory findings suggest a role for the outer BRB in the pathogenesis of diabetic retinopathy (DR). The aim of this study was to investigate whether the mechanisms involved in these alterations are sensitive to nitrosative stress, and if cocoa or epicatechin (EC) protects from this damage under diabetic (DM) milieu conditions. Cells of a human RPE line (ARPE-19) were exposed to high-glucose (HG) conditions for 24 hours in the presence or absence of cocoa powder containing 0.5% or 60.5% polyphenol (low-polyphenol cocoa [LPC] and high-polyphenol cocoa [HPC], respectively). Exposure to HG decreased claudin-1 and occludin TJ expressions and increased extracellular matrix accumulation (ECM), whereas levels of TNF-α and inducible nitric oxide synthase (iNOS) were upregulated, accompanied by increased nitric oxide levels. This nitrosative stress resulted in S-nitrosylation of caveolin-1 (CAV-1), which in turn increased CAV-1 traffic and its interactions with claudin-1 and occludin. This cascade was inhibited by treatment with HPC or EC through δ-opioid receptor (DOR) binding and stimulation, thereby decreasing TNF-α-induced iNOS upregulation and CAV-1 endocytosis. The TJ functions were restored, leading to prevention of paracellular permeability, restoration of resistance of the ARPE-19 monolayer, and decreased ECM accumulation. The detrimental effects on TJs in ARPE-19 cells exposed to DM milieu occur through a CAV-1 S-nitrosylation-dependent endocytosis mechanism. High-polyphenol cocoa or EC exerts protective effects through DOR stimulation.

Increased Circulating Pedf And Low Sicam-1 Are Associated With Sickle Cell Retinopathy.

Sickle cell retinopathy (SCR) develops in up to 30% of sickle cell disease patients (SCD) during the second decade of life. Treatment for this affection remains palliative, so studies on its pathophysiology may contribute to the future development of novel therapies. SCR is more frequently observed in hemoglobin SC disease and derives from vaso-occlusion in the microvasculature of the retina leading to neovascularization and, eventually, to blindness. Circulating inflammatory cytokines, angiogenic factors, and their interaction may contribute to the pathophysiology of this complication. Angiopoietin (Ang)-1, Ang-2, soluble vascular cell adhesion molecule-1, intercellular adhesion molecule (ICAM)-1, E-selectin, P-selectin, IL1-β, TNF-α, pigment epithelium derived factor (PEDF) and vascular endothelial growth factor plasmatic levels were determined in 37 SCD patients with retinopathy, 34 without retinopathy, and healthy controls. We observed that sICAM-1 is significantly decreased, whereas PEDF is elevated in HbSC patients with retinopathy (P=0.012 and P=0.031, respectively). Ang-1, Ang-2 and IL1-β levels were elevated in SCD patients (P=0.001, P<0.001 and P=0.001, respectively), compared to controls, and HbSS patients presented higher levels of Ang-2 compared to HbSC (P<0.001). Our study supports the possible influence of sICAM-1 and PEDF on the pathophysiology of retinal neovascularization in SCD patients.

Pigmented Ameloblastic Fibro-odontoma: Clinical, Histological, And Immunohistochemical Profile.

Ameloblastic fibro-odontoma (AFO) is a slow-growing, expansive, benign odontogenic tumor, composed of ameloblastic epithelium embedded in an ectomesenchymal stroma resembling dental papilla, containing hard dental tissue in variable degrees of maturation, including enamel, dentin, and sometimes cementum. AFO typically affects the posterior mandible, causing bony expansion. We report a case of pigmented AFO in a 5-year-old boy, comprising clinical and histological features illustrated by immunohistochemistry using a large panel of antibodies, polarized light microscopy and scanning electron microscopy.

Isolation of Pseudomonas aeruginosa strains from dental office environments and units in Barretos, state of São Paulo, Brazil, and analysis of their susceptibility to antimicrobial drugs

A wide variety of opportunistic pathogens has been detected in the tubing supplying water to odontological equipment, in special in the biofilm lining of these tubes. Among these pathogens, Pseudomonas aeruginosa, one of the leading causes of nosocomial infections, is frequently found in water lines supplying dental units. In the present work, 160 samples of water, and 200 fomite samples from forty dental units were collected in the city of Barretos, State of São Paulo, Brazil and evaluated between January and July, 2005. Seventy-six P. aeruginosa strains, isolated from the dental environment (5 strains) and water system (71 strains), were tested for susceptibility to six antimicrobial drugs most frequently used against P. aeruginosa infections. Susceptibility to ciprofloxacin, followed by meropenem was the predominant profile. The need for effective means of reducing the microbial burden within dental unit water lines is emphasized, and the risk of exposure and cross-infection in dental practice, in special when caused by opportunistic pathogens like P. aeruginosa, are highlighted.