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Acute ischemic stroke in the supply of the anterior inferior cerebellar artery is known to be connected with facial weakness, nystagmus, hearing loss, hypalgesia, and ataxia. This is a case of a young man with unilateral deafness caused due to infarction of anterior inferior cerebellar artery. Clinical findings and audiometry showed evidence suggestive of cochlear localization for deafness. MRI (Magnetic resonance imaging) Brain shows involvement of pons with infarct is seen in the right Anterior inferior cerebellar artery (AICA) territory. The inclusion of labyrinthine auditory artery suggests sensorineural hearing loss.

Anemia is a nearly universal diagnosis in preterm infants, caused by phlebotomy, and exacerbated by the underlying erythropoietic immaturity. Newborn infants are exposed to the unique stressor of fetal-to-neonatal transition, which requires significant adaptation ex utero. Accordingly, the preterm infant's response to anemia may alter the ability to confront underlying illness. This study utilized our preclinical mouse model of phlebotomy-induced anemia (PIA) to comprehensively investigate associated hematological changes.C57BL/6 mice were subjected to timed phlebotomy between postnatal days 2--10 to induce severe anemia. Complete blood counts were determined by the Sysmex XT-2000iV analyzer.Anemic pups showed a gradual reduction of RBC and hemoglobin (Hb) and increased reticulocyte (RET) counts and red cell distribution width (RDW), however, with reduced RET-Hb from postnatal day (P) of 4 onwards. Elevated levels of high fluorescent RET and immature reticulocyte fraction (IRF) were noted in anemic mouse pups, but low and medium fluorescent RET were reduced. Also, the reduction of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were noted in anemic pups. No changes were seen in lymphocytes, but monocytes and neutrophils were significantly elevated from P4-P6.PIA in mouse pups is associated with hematological changes that may be exacerbating factors in neonatal diseases.Anemia is common and often severe in premature infants. Investigation of hematological parameters in settings of preclinical anemia may be an index of therapeutic strategies. Preclinical model evaluating the effects of neonatal anemia on the remainder of complete blood count. Detailed time kinetic phlebotomy-induced anemic mice enable us to study the impact on developmental delays in erythropoiesis and possible strategic intervention. Hematological effects of severe anemia in mice might provide insight on how best to investigate anemia in preterm infants.

Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants and an orphan disease with no specific treatment. Most patients with confirmed NEC develop moderate-severe thrombocytopenia requiring one or more platelet transfusions. Here we used our neonatal murine model of NEC-related thrombocytopenia to investigate mechanisms of platelet depletion associated with this disease [K. Namachivayam, K. MohanKumar, L. Garg, B. A. Torres, A. Maheshwari, Pediatr. Res. 81, 817–824 (2017)]. In this model, enteral administration of immunogen trinitrobenzene sulfonate (TNBS) in 10-d-old mouse pups produces an acute necrotizing ileocolitis resembling human NEC within 24 h, and these mice developed thrombocytopenia at 12 to 15 h. We hypothesized that platelet activation and depletion occur during intestinal injury following exposure to bacterial products translocated across the damaged mucosa. Surprisingly, platelet activation began in our model 3 h after TNBS administration, antedating mucosal injury or endotoxinemia. Platelet activation was triggered by thrombin, which, in turn, was activated by tissue factor released from intestinal macrophages. Compared to adults, neonatal platelets showed enhanced sensitivity to thrombin due to higher expression of several downstream signaling mediators and the deficiency of endogenous thrombin antagonists. The expression of tissue factor in intestinal macrophages was also unique to the neonate. Targeted inhibition of thrombin by a nanomedicine-based approach was protective without increasing interstitial hemorrhages in the inflamed bowel or other organs. In support of these data, we detected increased circulating tissue factor and thrombin-antithrombin complexes in patients with NEC. Our findings show that platelet activation is an important pathophysiological event and a potential therapeutic target in NEC.

Anemia is a frequent diagnosis in critically ill infants, but the clinical implications of severe anemia in these patients remain unclear. In this study, we examined preweaned mice to investigate the effects of severe anemia during early infancy on gut mucosal permeability. C57BL/6 mice were subjected to timed phlebotomy between postnatal days (P) 2–10 to induce severe anemia (hematocrits 20%–24%), and intestinal permeability was tracked longitudinally between P10 and P20 as intestine-to-plasma translocation of enteral macromolecules and bacterial translocation. Epithelial junctions were evaluated by electron microscopy, polymerase chain reactions, immunohistochemistry, and/or enzyme immunoassays on intestinal tissues, Caco-2 intestinal epithelial-like cells, and colonic organoids. Preweaned mouse pups showed an age-related susceptibility to severe anemia, with increased intestinal permeability to enteral macromolecules (dextran, ovalbumin, β-lactoglobulin) and luminal bacteria. Electron micrographs showed increased paracellular permeability and ultrastructural abnormalities of the adherens junctions. These findings were explained by the loss of E-cadherin in epithelial cells, which was caused by destabilization of the E-cadherin ( Cdh1) mRNA because of microRNA let-7e-5p binding to the 3′-untranslated region. Severe anemia resulted in a disproportionate and persistent increase in intestinal permeability in preweaned mice because of the disruption of epithelial adherens junctions. These changes are mediated via microRNA let-7e-mediated depletion of Cdh1 mRNA. NEW & NOTEWORTHY This research article shows that newborn infants with severe anemia show an age-related susceptibility to developing increased intestinal permeability to ingested macromolecules. This abnormal permeability develops because of abnormalities in intestinal epithelial junctions caused by a deficiency of the molecule E-cadherin in epithelial cells. The deficiency of E-cadherin is caused by destabilization of its mRNA precursor because of increased expression and binding of another molecule, the microRNA let-7e-5p, to the E-cadherin mRNA.

Fetal swallowing of human amniotic fluid (hAF) containing trophic factors (TFs) promotes gastrointestinal tract (GIT) development. Preterm birth interrupts hAF swallowing, which may increase the risk of necrotizing enterocolitis (NEC). Postnatally, it is difficult to replicate fetal swallowing of hAF due to volume. We aimed to evaluate whether hAF lyophilization is feasible and its effect on hAF-borne TFs. We collected hAF (n = 16) from uncomplicated pregnancies. hAF was divided into three groups: unprocessed control (C), concentration by microfiltration (F), and by dialysis and lyophilization (L). EGF, HGF, GM-CSF, and TGF-α were measured in each group by multiplex assay. Bioavailability of TFs was measured by proliferation and LPS-induced IL-8 production by intestinal epithelial cells FHs74. After dialysis/lyophilization, GM-CSF and TGF-α were preserved with partial loss of EGF and HGF. hAF increased cell proliferation and reduced LPS-induced IL-8 production compared to medium alone. Compared to control, dialysis/lyophilization and filtration of hAF increased FHs74 cell proliferation (p

Inhibition of P300 acetyltransferase activity by specific inhibitor C646 has been shown to improve insulin signaling. However, the underlying molecular mechanism of this improvement remains unclear. In this study, we analyzed P300 levels of obese patients and found that they were significantly increased in liver hepatocytes. In addition, large amounts of P300 appeared in the cytoplasm. Inhibition of P300 acetyltransferase activity by C646 drastically increased tyrosine phosphorylation of the insulin receptor protein substrates (IRS1/2) without affecting the tyrosine phosphorylation of the beta subunit of the insulin receptor (IRβ) in hepatocytes in the absence of insulin. Since IRS1/2 requires membrane translocation and binding to inositol compounds for normal functions, we also examined the role of acetylation on binding to phosphatidylinositol(4,5)P2 and found that IRS1/2 acetylation by P300 reduced this binding. In contrast, we show that inhibition of IRS1/2 acetylation by C646 facilitates IRS1/2 membrane translocation. Intriguingly, we demonstrate that C646 activates IRβ's tyrosine kinase activity and directly promotes IRβ interaction with IRS1/2, leading to the tyrosine phosphorylation of IRS1/2 and subsequent activation of insulin signaling even in the absence of insulin. In conclusion, these data reveal the unique effects of C646 in activating insulin signaling in patients with obesity and diabetes.

Cytokines and growth factors play diverse roles in the uninflamed fetal/neonatal intestinal mucosa and in the development of inflammatory bowel injury during necrotizing enterocolitis (NEC). During gestational development and the early neonatal period, the fetal/premature intestine is exposed to high levels of many "inflammatory" cytokines and growth factors, first via swallowed amniotic fluid in utero and then, after birth, in colostrum and mother's milk. This article reviews the dual, seemingly counter-intuitive roles of cytokines, where these agents play a "trophic" role and promote maturation of the uninflamed mucosa, but can also cause inflammation and promote intestinal injury during NEC.

Background Thrombocytopenia is frequently encountered in infants with necrotizing enterocolitis (NEC). To develop a preclinical model of NEC-related thrombocytopenia, we measured serial platelet counts in 10-day-old (P10) mouse pups with trinitrobenzene sulfonic acid (TNBS)-induced NEC-like injury. We also measured platelet volume indices, immature platelet fraction (IPF), and megakaryocyte number/ploidy in these animals. Methods Platelet counts, platelet volume indices, and IPF were measured in control (N=65) and TNBS-treated pups (N=104) using an automated hematology analyzer. Bone marrow megakaryocyte number, ploidy and CD41 expression were measured by flow cytometry. These findings were confirmed in a small cohort of P3 mice with NEC-like injury. Results Murine pups with TNBS-mediated NEC-like injury developed thrombocytopenia at 15–24h after exposure to TNBS. Intestinal injury was associated with increased platelet volume indices (mean platelet volume, platelet-to-large cell ratio, and platelet distribution width), and IPF, indicating increased thrombopoiesis. These mice also showed increased megakaryocyte number, ploidy, and CD41 expression, indicating increased megakaryocyte differentiation. Conclusions Similar to human NEC, murine NEC-like injury was also associated with decreased platelet counts. There was evidence of increased megakaryocyte differentiation and thrombopoiesis, which favors peripheral consumption of platelets as the likely mechanism of thrombocytopenia in these animals, over decreased platelet production.

We have shown previously that enteral administration of 2, 4, 6-trinitrobenzene sulfonic acid in 10-d-old C57BL/6 pups produces an acute necrotizing enterocolitis with histopathological and inflammatory changes similar to human necrotizing enterocolitis (NEC). To determine whether murine neonatal 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-mediated intestinal injury could be used as a NEC model, we compared gene expression profiles of TNBS-mediated intestinal injury and NEC.Whole-genome microarray analysis was performed on proximal colon from control and TNBS-treated pups (n = 8/group). For comparison, we downloaded human microarray data of NEC (n = 5) and surgical control (n = 4) from a public database. Data were analyzed using the software programs Partek Genomics Suite and Ingenuity Pathway Analysis.We detected extensive changes in gene expression in murine TNBS-mediated intestinal injury and human NEC. Using fold-change cut-offs of ±1.5, we identified 4,440 differentially-expressed genes (DEGs) in murine TNBS-mediated injury and 1,377 in NEC. Murine TNBS-mediated injury and NEC produced similar changes in expression of orthologous genes (r = 0.611, P0.001), and also activated nearly-identical biological processes and pathways. Lipopolysaccharide was top predicted upstream regulator in both the murine and human datasets.Murine neonatal TNBS-mediated enterocolitis and human NEC activate nearly-identical biological processes, signaling pathways, and transcriptional networks.

Background Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. Based on our recent findings of increased Smad7 expression in surgically-resected bowel affected by NEC, we hypothesized that NEC macrophages undergo inflammatory activation because increased Smad7 expression renders these cells resistant to normal, gut-specific, transforming growth factor (TGF)-β-mediated suppression of inflammatory pathways. Methods We used surgically-resected human NEC tissue, murine models of NEC-like injury, bone marrow-derived and intestinal macrophages, and RAW264.7 cells. Smad7 and IκB kinase-beta (IKK-β) were measured by quantitative polymerase chain reaction (qPCR), Western blots, and immunohistochemistry. Promoter activation was confirmed in luciferase reporter and chromatin immunoprecipitation assays. Results NEC macrophages showed increased Smad7 expression, particularly in areas with severe tissue damage and high bacterial load. LPS-induced Smad7 expression suppressed TGF-β signaling and augmented NF-κB activation and cytokine production in macrophages. Smad7-mediated NF-κB activated was likely mediated via increased expression of IKK-β, which, further increased Smad7 expression in a feed-forward loop. We show that Smad7 induced IKK-β expression through direct binding to the IKK-β promoter and its transcriptional activation. Conclusions Smad7 expression in NEC macrophages interrupts TGF-β signaling and promotes NF-κB-mediated inflammatory signaling in these cells through increased expression of IKK-β.

Vascular endothelial growth factor (VEGF), a well-characterized regulator of angiogenesis, has been mechanistically implicated in retinal neovascularization and in the pathogenesis of retinopathy of prematurity. However, the ontogeny of VEGF expression in the human fetal retina is not well known. Because retinal vasculature grows with gestational maturation, we hypothesized that VEGF expression also increases in the midgestation human fetal eye as a function of gestational age. To identify changes in VEGF gene expression during normal human development, we measured VEGF mRNA by quantitative PCR and measured VEGF protein by enzyme-linked immunosorbent assay and western blots in 10–24 wk gestation fetal vitreous, retina, and serum. VEGF mRNA expression in the retina increased with gestational age. VEGF isoform A, particularly its VEGF121 splice variant, contributed to this positive correlation. Consistent with these findings, we detected increasing VEGF121 protein concentrations in vitreous humor from fetuses of 10–24 wk gestation, while VEGF concentrations decreased in fetal serum. VEGF121 mRNA and protein concentrations increase with increasing gestational age in the developing human retina. We speculate that VEGF plays an important role in normal retinal vascular development, and that preterm delivery affects production of this vascular growth factor.

Background Neonates and young infants exposed to extracorporeal circulation during extracorporeal membrane oxygenation (ECMO) and cardiopulmonary bypass (CPB) are at risk of developing a systemic inflammatory response syndrome (SIRS) with multi-organ dysfunction. We used a piglet model of ECMO to investigate the hypothesis that epithelial apoptosis is an early event that precedes villous damage during ECMO-related bowel injury. Methods Healthy 3-week-old piglets were subjected to ECMO for up to 8h. Epithelial apoptosis was measured in histopathological analysis, nuclear imaging, and terminal deoxynucleotidyl transferase dUTP nick end labeling. Plasma intestinal-fatty acid-binding protein (I-FABP) levels were measured by enzyme immunoassay. Intestinal mast cells were isolated by fluorescence-assisted cell sorting. Cleaved caspase-8, caspase-9, phospho-p38 MAPK, and fas ligand expression was investigated by immunohistochemistry, Western blots, and reverse transcriptase-quantitative polymerase chain reaction. Results Piglet ECMO was associated with increased gut epithelial apoptosis. Extensive apoptotic changes were noted on villus tips and in scattered crypt cells after 2h of ECMO. After 8h, the villi were denuded and apoptotic changes were evident in a majority of crypt cells. Increased circulating I-FABP levels, a marker of gut epithelial injury, showed that epithelial injury occurred during ECMO. We detected increased cleaved caspase-8, but not cleaved caspase-9, in epithelial cells indicating that the extrinsic apoptotic pathway was active. ECMO was associated with increased fas ligand expression in intestinal mast cells, which was induced through activation of the p38 mitogen-activated protein kinase. Conclusions Epithelial apoptosis is an early event that initiates gut mucosal injury in a piglet model of ECMO.

Human milk contains substantial amounts of transforming growth factor (TGF)-β, particularly the isoform TGF-β2. We previously showed in preclinical models that enterally administered TGF-β2 can protect against necrotizing enterocolitis (NEC), an inflammatory bowel necrosis of premature infants. In this study we hypothesized that premature infants remain at higher risk of NEC than full-term infants, even when they receive their own mother's milk, because preterm human milk contains less bioactive TGF-β than full-term milk. Our objective was to compare TGF-β bioactivity in preterm vs. full-term milk and identify factors that activate milk-borne TGF-β. Mothers who delivered between 23 0/7 and 31 6/7 wk or at ≥37 wk of gestation provided milk samples at serial time points. TGF-β bioactivity and NF-κB signaling were measured using specific reporter cells and in murine intestinal tissue explants. TGF-β1, TGF-β2, TGF-β3, and various TGF-β activators were measured by real-time PCR, enzyme immunoassays, or established enzymatic activity assays. Preterm human milk showed minimal TGF-β bioactivity in the native state but contained a large pool of latent TGF-β. TGF-β2 was the predominant isoform of TGF-β in preterm milk. Using a combination of several in vitro and ex vivo models, we show that neuraminidase is a key regulator of TGF-β bioactivity in human milk. Finally, we show that addition of bacterial neuraminidase to preterm human milk increased TGF-β bioactivity. Preterm milk contains large quantities of TGF-β, but most of it is in an inactive state. Addition of neuraminidase can increase TGF-β bioactivity in preterm milk and enhance its anti-inflammatory effects.

Cancer is a leading cause of deaths in the world, with lung, breast and colorectal cancers being the top killers. As early detection is important to prevent deaths due to cancer, current emphasis is on periodic screening for persons over the age of 50. Depending on the organ being assessed, a variety of diagnostics tools are presently available to detect abnormal state of the tissue, with each technique having its own set of advantages and disadvantages. Of these, currently, only endoscopy provides a possibility of abnormality detection based on direct tissue imaging and visual differentiation in body cavities such as colon. However, the effectiveness of endoscopy is limited in that polyps are missed as they fail to catch the attention of the endoscopist during the visual examination. They are missed out in blind biopsies or are not well delineated as abnormalities, especially as in the case of flat dysplasia. If abnormal regions are well identified, targeted biopsies can be carried out minimising the pain and risk of tissue perforations to the patient. Research is in progress around the world for finding methods which are minimally invasive and which can provide accurate results with minimum patient discomfort. The hallmarks of cancer are changes in the tissue’s biochemical makeup, biological activity level, vasculature, blood perfusion and cellular structure. In this context, this research thesis focuses on coming up with such methodologies using optical concepts and imaging modalities for diagnostic medical imaging in a probe based scheme. Complementary optical methodologies, which can enable easy identification and diagnosis of potential abnormal growth sites that represent cancerous state of tissue, were investigated in this research, as it is envisioned that information from one modality can be used to strengthen judgment from another independent modality. For the modalities in whole field imaging mode, a target size of 5 mm and below was set as detection capability for the abnormalities for early diagnosis, as polyp sizes up to 5 mm were deemed as low risk and are only marked for observation in subsequent screening sessions. An enlargement of the size of cell nucleus from about 5-10 pm to about 20 pm being an indication of pre-cancerous state, a resolution capability of less than 5 pm was set as a target for high resolution imaging mode. A novel RGB grayscale value based spectral shift approach was successfully developed for easy identification of abnormality based on biochemical signature differences that can be identified optically, namely fluorescence. A CCD camera based image analysis scheme was conceptualised and configured using spectral shift calculations. The Euclidian distances for the change in Red, Green and Blue gray values between the illuminating wavelengths and the points in the image were used to reconstruct a pseudo-image which shows the differences between normal and abnormal areas in the tissues. The proposed algorithm was validated with phantom tissues as well as real mouse colon tissues and mouse with tumour to distinguish abnormal regions, with abnormality sizes ranging from 2 mm to about 12 mm. The proposed algorithm and developed methodology apply pixel level processing, and hence, processing of both whole field and high resolution images can be carried out.Contouring of abnormality based on bioactivity differences that can be identified optically, namely dynamic speckle based methodology, was the second target area of the research. A novel thermal perturbation based approach was proposed and researched to enable identification and contouring of abnormalities by way of differences in speckle pattern changes arising out of tissue mass and perfusion differences. Thermal perturbation of the target tissue with infrared beam was used to effect the required change in the bio-speckle pattern. Subsequent contouring of the abnormal region in the tissue could be delineated due to the speckle pattem differences that evolved over. It was found, using phantom tissue as test samples, that a time lapse of about 1 second between imaging frames captured with a CCD camera could enable identification and contouring of sub-surface abnormalities. An image reconstruction scheme using variation of intensity at corresponding pixel locations was also investigated. The effects of perfusion and simulated mucus layer were investigated. Experimental verification was carried out on silicone phantoms and to a limited extent, with animal model, with abnormality sizes ranging from about 5 mm to 8 mm. A fiber based high resolution imaging methodology was investigated that is capable of producing microscopic images of the targeted tissues. A quasi- confocal imaging approach with an imaging fiber bundle was developed to image and contour microscale objects. Non-scanning (fixed frame) as well as scanning (lD as well as 2D) based approaches were investigated. For the nonscan based approach, with axial and lateral resolutions of 15.98 ± 0.94 pm and 3.47 pm, respectively, the system capability was demonstrated with glassbubbles, polystyrene beads, cells and micro-fluidic flow channels. The scan based methodology developed, with the probe in proximity with the target tissue, could overcome loss of visual information due to the fiberlet interspaces and achieved a significant improvement in lateral resolution to 2.19 pm with the same optical elements as with the non-scan based scheme. With the fiber bundle enabling multiple scan points, thus saving scan time, and scanning steps designed to match the imaging pixel size for best resolution, a scheme to reconstruct high resolution images from the whole-field scan, at any region of interest was worked out. It was found that the central pixel in each of the fiberlet image are the optimal scan points as it gave the best contrast and the least offset in the scan image compared with other pixels within the fiberlet. The capability of the system was demonstrated using micron-sized fluorescent polystyrene beads and cells mounted on a microscope slide. Besides providing imaging capability, the imaging fibre can double up as a light conduit to illuminate the target tissue, which would be in synergy with the first two modalities, namely, the RGB and the dynamic speckle analyses. The research carried out in this thesis involved exploring and laying the foundations for a synergistic combination of optical methods which could potentially be integrated in future into a single probe for endoscopic applications. It is capable of identifying early stage cancerous regions of less than 5 mm in size from a distance and also micro-level imaging of structures less than 5 pm at proximity of the targeted area. As a future work direction, an actual probe system could be engineered integrating a micro-CCD camera and an imaging fiber bundle by applying the proposed multiple optical bubbles, polystyrene beads, cells and micro-fluidic flow channels. The scan based methodology developed, with the probe in proximity with the target tissue, could overcome loss of visual information due to the fiberlet interspaces and achieved a significant improvement in lateral resolution to 2.19 pm with the same optical elements as with the non-scan based scheme. With the fiber bundle enabling multiple scan points, thus saving scan time, and scanning steps designed to match the imaging pixel size for best resolution, a scheme to reconstruct high resolution images from the whole-field scan, at any region of interest was worked out. It was found that the central pixel in each of the fiberlet image are the optimal scan points as it gave the best contrast and the least offset in the scan image compared with other pixels within the fiberlet. The capability of the system was demonstrated using micron-sized fluorescent polystyrene beads and cells mounted on a microscope slide. Besides providing imaging capability, the imaging fibre can double up as a light conduit to illuminate the target tissue, which would be in synergy with the first two modalities, namely, the RGB and the dynamic speckle analyses. The research carried out in this thesis involved exploring and laying the foundations for a synergistic combination of optical methods which could potentially be integrated in future into a single probe for endoscopic applications. It is capable of identifying early stage cancerous regions of less than 5 mm in size from a distance and also micro-level imaging of structures less than 5 pm at proximity of the targeted area. As a future work direction, an actual probe system could be engineered integrating a micro-CCD camera and an imaging fiber bundle by applying the proposed multiple optical bubbles, polystyrene beads, cells and micro-fluidic flow channels. The scan based methodology developed, with the probe in proximity with the target tissue, could overcome loss of visual information due to the fiberlet interspaces and achieved a significant improvement in lateral resolution to 2.19 pm with the same optical elements as with the non-scan based scheme. With the fiber bundle enabling multiple scan points, thus saving scan time, and scanning steps designed to match the imaging pixel size for best resolution, a scheme to reconstruct high resolution images from the whole-field scan, at any region of interest was worked out. It was found that the central pixel in each of the fiberlet image are the optimal scan points as it gave the best contrast and the least offset in the scan image compared with other pixels within the fiberlet. The capability of the system was demonstrated using micron-sized fluorescent polystyrene beads and cells mounted on a microscope slide. Besides providing imaging capability, the imaging fibre can double up as a light conduit to illuminate the target tissue, which would be in synergy with the first two modalities, namely, the RGB and the dynamic speckle analyses. The research carried out in this thesis involved exploring and laying the foundations for a synergistic combination of optical methods which could potentially be integrated in future into a single probe for endoscopic applications. It is capable of identifying early stage cancerous regions of less than 5 mm in size from a distance and also micro-level imaging of structures less than 5 pm at proximity of the targeted area. As a future work direction, an actual probe system could be engineered integrating a micro-CCD camera and an imaging fiber bundle by applying the proposed multiple optical methodologies for effective identification, contouring and confirmation of abnormalities that can lead to early diagnosis of disease, such as cancer, in body cavities such as colon. These findings are expected to contribute to future research towards realising an in vivo optical biopsy probe. Doctor of Philosophy (MAE)

Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. In tissue samples of NEC, we identified numerous macrophages and a few neutrophils but not many lymphocytes. We hypothesized that these pathoanatomic characteristics of NEC represent a common tissue injury response of the gastrointestinal tract to a variety of insults at a specific stage of gut development. To evaluate developmental changes in mucosal inflammatory response, we used trinitrobenzene sulfonic acid (TNBS)-induced inflammation as a nonspecific insult and compared mucosal injury in newborn vs. adult mice. Enterocolitis was induced in 10-day-old pups and adult mice ( n = 25 animals per group) by administering TNBS by gavage and enema. Leukocyte populations were enumerated in human NEC and in murine TNBS-enterocolitis using quantitative immunofluorescence. Chemokine expression was measured using quantitative polymerase chain reaction, immunoblots, and immunohistochemistry. Macrophage recruitment was investigated ex vivo using intestinal tissue-conditioned media and bone marrow-derived macrophages in a microchemotaxis assay. Similar to human NEC, TNBS enterocolitis in pups was marked by a macrophage-rich leukocyte infiltrate in affected tissue. In contrast, TNBS-enterocolitis in adult mice was associated with pleomorphic leukocyte infiltrates. Macrophage precursors were recruited to murine neonatal gastrointestinal tract by the chemokine CXCL5, a known chemoattractant for myeloid cells. We also demonstrated increased expression of CXCL5 in surgically resected tissue samples of human NEC, indicating that a similar pathway was active in NEC. We concluded that gut mucosal injury in the murine neonate is marked by a macrophage-rich leukocyte infiltrate, which contrasts with the pleomorphic leukocyte infiltrates in adult mice. In murine neonatal enterocolitis, macrophages were recruited to the inflamed gut mucosa by the chemokine CXCL5, indicating that CXCL5 and its cognate receptor CXCR2 merit further investigation as potential therapeutic targets in NEC.

Imaging of micro particles in micro-fluidic channels is one of the recent thrust areas in research as it provides more flexibility in setup and analysis compared to conventional microscopy. However, a probe based imaging scheme, with achievable high resolutions incorporating multimodal analysis is one of the challenges researchers have been facing. In this context, this paper illustrates a simple dual modality high resolution flexible probe imaging system for imaging applications in micro/optofluidic channels. The proposed system exhibits axial and lateral resolution of about 16 μm and 3.12 μm respectively. This proposed system also exhibits a modulation transfer function (MTF) of about 38.42 %. The performance of the system is validated by imaging micro particles in a microchannel and obtaining the fluorescent emission spectrum simultaneously.

Objective We have shown previously that preterm infants are at risk of necrotizing enterocolitis (NEC), an inflammatory bowel necrosis typically seen in infants born prior to 32 weeks’ gestation, because of the developmental deficiency of transforming growth factor (TGF)-β2 in the intestine. The present study was designed to investigate all-trans retinoic acid (atRA) as an inducer of TGF-β2 in intestinal epithelial cells (IECs) and to elucidate the involved signaling mechanisms. Methods AtRA effects on intestinal epithelium were investigated using IEC6 cells. TGF-β2 expression was measured using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Western blots. Signaling pathways were investigated using Western blots, transiently-transfected/transduced cells, kinase arrays, chromatin immunoprecipitation, and selective small molecule inhibitors. Results AtRA-treatment of IEC6 cells selectively increased TGF-β2 mRNA and protein expression in a time- and dose-dependent fashion, and increased the activity of the TGF-β2 promoter. AtRA effects were mediated via RhoA GTPase, Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), p38α MAPK, and activating transcription factor (ATF)-2. AtRA increased phospho-ATF2 binding to the TGF-β2 promoter and increased histone H2B acetylation in the TGF-β2 nucleosome, which is typically associated with transcriptional activation. Conclusions AtRA induces TGF-β2 expression in IECs via RhoA- and p38α MAPK-mediated activation of the transcription factor ATF2. Further studies are needed to investigate the role of atRA as a protective/therapeutic agent in gut mucosal inflammation.

Human milk contains biologically important amounts of transforming growth factor-β2isoform (TGF-β2), which is presumed to protect against inflammatory gut mucosal injury in the neonate. In preclinical models, enterally administered TGF-β2can protect against experimental necrotizing enterocolitis, an inflammatory bowel necrosis of premature infants. In this study, we investigated whether TGF-β bioactivity in human preterm milk could be enhanced for therapeutic purposes by adding recombinant TGF-β2(rTGF-β2) to milk prior to feeding. Milk-borne TGF-β bioactivity was measured by established luciferase reporter assays. Molecular interactions of TGF-β2were investigated by nondenaturing gel electrophoresis and immunoblots, computational molecular modeling, and affinity capillary electrophoresis. Addition of rTGF-β2(20–40 nM) to human preterm milk samples failed to increase TGF-β bioactivity in milk. Milk-borne TGF-β2was bound to chondroitin sulfate (CS) containing proteoglycan(s) such as biglycan, which are expressed in high concentrations in milk. Chondroitinase treatment of milk increased the bioactivity of both endogenous and rTGF-β2, and consequently, enhanced the ability of preterm milk to suppress LPS-induced NF-κB activation in macrophages. These findings provide a mechanism for the normally low bioavailability of milk-borne TGF-β2and identify chondroitinase digestion of milk as a potential therapeutic strategy to enhance the anti-inflammatory effects of preterm milk.

Fetal swallowing of amniotic fluid, which contains numerous cytokines and growth factors, plays a key role in gut mucosal development. Preterm birth interrupts this exposure to amniotic fluid-borne growth factors, possibly contributing to the increased risk of necrotizing enterocolitis (NEC) in premature infants. We hypothesized that supplementation of formula feeds with amniotic fluid can provide amniotic fluid-borne growth factors and prevent experimental NEC in rat pups. We compared NEC-like injury in rat pups fed with infant formula vs. formula supplemented either with 30% amniotic fluid or recombinant hepatocyte growth factor (HGF). Cytokines/growth factors in amniotic fluid were measured by immunoassays. Amniotic fluid and HGF effects on enterocyte migration, proliferation, and survival were measured in cultured IEC6 intestinal epithelial cells. Finally, we used an antibody array to investigate receptor tyrosine kinase (RTK) activation and immunoblots to measure phosphoinositide 3-kinase (PI3K) signaling. Amniotic fluid supplementation in oral feeds protected rat pups against NEC-like injury. HGF was the most abundant growth factor in rat amniotic fluid in our panel of analytes. Amniotic fluid increased cell migration, proliferation, and cell survival in vitro. These effects were reproduced by HGF and blocked by anti-HGF antibody or a PI3K inhibitor. HGF transactivated several RTKs in IEC6 cells, indicating that its effects extended to multiple signaling pathways. Finally, similar to amniotic fluid, recombinant HGF also reduced the frequency and severity of NEC-like injury in rat pups. Amniotic fluid supplementation protects rat pups against experimental NEC, which is mediated, at least in part, by HGF.

Preterm infants may be at risk of necrotizing enterocolitis (NEC) due to deficiency of transforming growth factor-β 2 (TGF-β2) in the developing intestine. We hypothesized that low epithelial TGF-β2expression in preterm intestine and during NEC results from diminished autocrine induction of TGF-β2in these cells. Premature baboons delivered at 67% gestation were treated per current norms for human preterm infants. NEC was diagnosed by clinical and radiological findings. Inflammatory cytokines, TGF-β2, Smad7, Ski, and strawberry notch N (SnoN)/Ski-like oncoprotein (SKIL) was measured using quantitative reverse transcriptase-polymerase chain reaction, immunoblots, and immunohistochemistry. Smad7 effects were examined in transfected IEC6 intestinal epithelial cells in vitro. Findings were validated in archived human tissue samples of NEC. NEC was recorded in seven premature baboons. Consistent with existing human data, premature baboon intestine expressed less TGF-β2than term intestine. TGF-β2expression was regulated in epithelial cells in an autocrine fashion, which was interrupted in the premature intestine and during NEC due to increased expression of Smad7. LPS increased Smad7 binding to the TGF-β2promoter and was associated with dimethylation of the lysine H3K9, a marker of transcriptional silencing, on the nucleosome of TGF-β2. Increased Smad7 expression in preterm intestine was correlated with the deficiency of SnoN/SKIL, a repressor of the Smad7 promoter. Smad7 inhibits autocrine expression of TGF-β2in intestinal epithelial cells in the normal premature intestine and during NEC. Increased Smad7 expression in the developing intestine may be due to a developmental deficiency of the SnoN/SKIL oncoprotein.

Chlorine gas (Cl(2)) exposure during accidents or in the military setting results primarily in injury to the lungs. However, the potential for Cl(2) exposure to promote injury to the systemic vasculature leading to compromised vascular function has not been studied. We hypothesized that Cl(2) promotes extrapulmonary endothelial dysfunction characterized by a loss of endothelial nitric oxide synthase (eNOS)-derived signaling. Male Sprague Dawley rats were exposed to Cl(2) for 30 minutes, and eNOS-dependent vasodilation of aorta as a function of Cl(2) dose (0-400 ppm) and time after exposure (0-48 h) were determined. Exposure to Cl(2) (250-400 ppm) significantly inhibited eNOS-dependent vasodilation (stimulated by acetycholine) at 24 to 48 hours after exposure without affecting constriction responses to phenylephrine or vasodilation responses to an NO donor, suggesting decreased NO formation. Consistent with this hypothesis, eNOS protein expression was significantly decreased (∼ 60%) in aorta isolated from Cl(2)-exposed versus air-exposed rats. Moreover, inducible nitric oxide synthase (iNOS) mRNA was up-regulated in circulating leukocytes and aorta isolated 24 hours after Cl(2) exposure, suggesting stimulation of inflammation in the systemic vasculature. Despite decreased eNOS expression and activity, no changes in mean arterial blood pressure were observed. However, injection of 1400W, a selective inhibitor of iNOS, increased mean arterial blood pressure only in Cl(2)-exposed animals, suggesting that iNOS-derived NO compensates for decreased eNOS-derived NO. These results highlight the potential for Cl(2) exposure to promote postexposure systemic endothelial dysfunction via disruption of vascular NO homeostasis mechanisms.

We have developed a variable focal lengths image fiber based microscope system for biomedical application. The objective lens of the proposed system comprises lens of variable focal lengths and is prepared on micro glass slide by spin coating technique. The position of each lens is aligned in precision along the optical axis of system by employing high resolution xyz stage. The performance of the system is verified by imaging fluorescent microspheres of size ∼ 1μm. The proposed system can be employed in real-time fiber based high resolution imaging for biological specimens.

High-resolution measurement and contouring of objects with micro-scale sizes have been two of the research challenges in many areas, such as nondestructive testing and imaging, imaging of artifacts in MEMS, and lab-on-chip devices, as well as in biomedical imaging. In this context, we use a transillumination incorporated fiber probe imaging system to enable imaging of the targeted object in a single shot. It also enables positioning of the probe system to the region of interest for further fine analysis, thereby reducing the long scanning time faced by conventional approaches. The capability of the developed probe is illustrated using standard USAF resolution chart and fluorescent microspheres as test targets. The probe system has axial and lateral resolutions of about 16 μm and 144 lp/mm, respectively. This proposed probe scheme can potentially be employed as a viable diagnostic imaging methodology.