Androgen Receptor Expression in Hypospadias.

INTRODUCTION: The exact mechanism behind the development of hypospadias is unclear. Research studies on androgen receptor (AR) expression are controversial with results stating all possible outcomes - AR elevated, similar, or reduced when compared to normal. AIMS: The aim is to study the AR expression and hormone levels in hypospadias patients and compare them with children having normal genitalia. METHODS: Group 1 (controls) involved patients who underwent circumcision for phimosis while Group 2 involved hypospadias patients who did not receive any preoperative testosterone. Preoperative hormonal assay included luteinizing hormone, follicle-stimulating hormone, and free testosterone levels in all the patients. The foreskin specimen was analyzed for AR expression using immunohistochemistry (anti-AR antibody PathnSitu, clone R441, 1/100 dilution). AR staining was expressed as H score. The H score was calculated by multiplying the intensity of staining and the percentage of stained cells showing cytoplasmic positivity at high power (×40). RESULTS: There were 27 patients in Group 1 while 16 in Group 2 (distal 10; proximal 6).There was no significant difference in the age distribution. The mean H score was significantly higher (189.5) in hypospadias patients compared to controls (97.5) and was significantly higher in proximal (220) compared to distal (159) hypospadias. There was no significant difference in hormone levels between groups. CONCLUSION: AR expression was significantly elevated in hypospadias patients. It was higher in proximal compared to distal hypospadias, probably due to end-organ overexpression. Further larger trials are likely to through light into this controversial subject.

Cytotoxicity of hepatocellular carcinoma cells to hyperthermic and ablative temperature exposures: in vitro studies and mathematical modelling.

PURPOSE: Image-guided ablative therapies use temperatures greater than 45 °C to kill abnormal cells. There is limited published data of cell survival after ablative temperature exposures, which is of importance to predict ablation zone dimensions. The objective of this study was to determine and mathematically model survival of hepatocellular carcinoma cells following ablative temperature exposures (45-60 °C). MATERIALS AND METHODS: Hepatocellular carcinoma (HCC) cell lines were plated in 96-well plates, and heated between 45 and 60 °C for 0-32 min. Heating was applied by a rapid media exchange with heated Hank's balanced salt solution (HBSS) in a temperature-controlled water bath. Cell viability was determined by MTS assay. Survival data was modelled by the Arrhenius model, and the thermal isoeffective dose (TID) model where kinetic parameters were determined via non-linear optimisation. RESULTS: Results suggest that the thermal dose based on cumulative equivalent minutes and parameters as used for hyperthermia exposures (<43 °C) is not applicable for ablative exposures. We found R = 0.72 for temperatures between 45-60°C for the TID model. The Arrhenius parameters were frequency factor A = 3.25E43 1/s, and activation energy Ea = 281 kJ/mol. These parameters correlate well with a prior study in the same cell line, and with threshold temperatures for necrosis from in vivo studies. CONCLUSIONS: Our results suggest that standard TID model kinetic parameters based on hyperthermia studies, often also used at ablation temperatures, are not applicable at these higher temperatures for HCC cells.

An evaluation of dentofacial changes in Angle's class II division 1 patients using AdvanSync 2.

OBJECTIVES: The purpose of the study is as follows: To evaluate the dental and skeletal changes of the AdvanSync 2 appliance.To evaluate the soft tissue changes of the AdvanSync 2 appliance using photometric analysis. METHODS: The sample size consisted of 15 patients who reported to the Department of Orthodontics, seeking fixed orthodontic treatment. The effects of the AdvanSync 2 appliance were measured at two intervals. RESULTS: After the nine months, P values were observed to be less than 0.5, therefore statistically significant for parameters such as Sella-Nasion-Point A (SNA), Condylion-Point A (CO-A), University of Witwatersrand, Condylion-Gnathion (C0-Gn), point A-Nasion-Point B (ANB), Upper incisor-Point A (UI-A) (degree), LI-B (mm), Lower lip to Esthetic plane (LL-E plane), nasolabial angle, mentolabial angle, facial angle, and L lip to the chin. P values were however observed to be greater than 0.5, therefore statistically insignificant for parameters such as sella-Nasion-Point B (SNB), Condylion -gonion (C0-Go), UI A (mm), LI B (mm), UL-EPL, H LINE, Frankfurt mandibular plane (FMA), nose tip angle, nasofrontal angle, nasomental angle, upper lip angle, and U lip to the chin. CONCLUSIONS: AdvanSync 2 appliance brought about a change in Class II malocclusions through Co-Gn, Co-Go, ANB, FMA, UI-A (degree), UI A (linear) LI B (linear), Upper lip to Esthetic plane, LL-E plane, Holdaway LINE, nose tip angle, nasolabial angle, mentolabial angle, nasofrontal angle, nasomental angle, facial angle, upper lip angle, U lip to chin, L lip to the chin after nine months of appliance delivery. MAIN POINTS: AdvanSync 2 normalized class II by an increase in the length and body of the mandible.AdvanSync 2 has a restraining effect on the growth of the maxilla.AdvanSync 2 brings about positive soft tissue changes.The major disadvantage is the proclination of the lower incisors.

Radiopaque drug-eluting beads for transcatheter embolotherapy: experimental study of drug penetration and coverage in swine.

PURPOSE: To determine local doxorubicin levels surrounding radiopaque drug-eluting beads (DEBs) in normal swine liver and kidney following transcatheter arterial chemoembolization. The influence of bead size (70-150 µm or 100-300 µm) was compared with regard to tissue penetration and spatial distribution of the bead, as well as eventual drug coverage (ie, amount of tissue exposed to drug). MATERIALS AND METHODS: Radiopaque DEBs were synthesized by suspension polymerization followed by incorporation of iodized oil and doxorubicin. Chemoembolization of swine liver and kidney was performed under fluoroscopic guidance. Three-dimensional tissue penetration of "imageable" DEBs was investigated ex vivo with micro-computed tomography (microCT). Drug penetration from the bead surface and drug coverage was evaluated with epifluorescence microscopy, and cellular localization of doxorubicin was evaluated with confocal microscopy. Necrosis was evaluated with hematoxylin and eosin staining. RESULTS: MicroCT demonstrated that 70-150-µm DEBs were present in more distal arteries and located in a more frequent and homogeneous spatial distribution. Tissue penetration of doxorubicin from the bead appeared similar (∼300 µm) for both DEBs, with a maximum tissue drug concentration at 1 hour coinciding with nuclear localization of doxorubicin. The greater spatial frequency of the 70-150-µm DEBs resulted in approximately twofold improved drug coverage in kidney. Cellular death is predominantly observed around the DEBs beginning at 8 hours, but increased at 24 and 168 hours. CONCLUSIONS: Smaller DEBs penetrated further into targeted tissue (ie, macroscopic) with a higher spatial density, resulting in greater and more uniform drug coverage (ie, microscopic) in swine.

Comparative assessment of changes in pharyngeal airway space in cases of obstructive sleep apnoea with a customized mandibular repositioning appliance - a clinical study.

OBJECTIVES: The present study aims at evaluating the effects of a customized mandibular repositioning appliance on the pharyngeal airway, nocturnal sleep patterns, daytime discomfort and occlusal changes in established cases of adult obstructive sleep apnoea. MATERIAL AND METHODS: Ten consecutive patients with a complaint of snoring and disturbed sleep were included in the study. The primary diagnosis was established by the Epworth sleepiness scale, clinical examination, history and subsequently the diagnosis was substantiated through assessment of the pharyngeal airway space on a lateral cephalogram and polysomnography. A customized mandibular repositioning appliance was used to advance the mandible sequentially every 6 months, using 4 sets of the appliance. Pre and post-treatment evaluations were performed to establish, effects and changes in the outcome of obstructive sleep apnoea. RESULTS: The study revealed significant increase in the mean pharyngeal widths of upper airway and velum dimension with antero-superior repositioning of hyoid bone. Epworth sleepiness scale score improved significantly from baseline with clinically evident change in daytime discomforts. Significant decline in the mean apnoea/hypopnea index, oxygen desaturation index, respiratory disturbance index, heart rate, snoring and a significant increase in mean oxygen saturation of arterial blood was observed. No evident change noticed in occlusion except lower incisor inclination. CONCLUSION: The customized mandibular repositioning appliances are effective in the management of adult obstructive sleep apnoea with a significant improvement observed in the airway patency and polysomnography parameters with clinically non-significant effects on dental occlusion..

A Comparison of the Rate of Retraction with Low-level Laser Therapy and Conventional Retraction Technique.

BACKGROUND AND OBJECTIVES: A major concern of orthodontic patients is treatment time. Reducing the treatment time requires increasing the rate of orthodontic tooth movement. Research has proved that bone resorption is the rate-limiting step in tooth movement. Therefore, any procedure that potentiates osteoclastic activity is capable of increasing the rate of orthodontic tooth movement. Low-level laser has been indicated to have the capability to facilitate the differentiation of the osteoclastic and osteoblastic cells, which are responsible for the bone remodeling process. The purpose of this study was to evaluate whether the low-level laser therapy can accelerate orthodontic tooth movement during en masse retraction. METHOD: The study was a split-mouth design. The experimental side was exposed to biostimulation using 810 nm gallium-aluminium-arsenide diode laser. A total of 10 irradiations for 10 s per site were given 5 on the buccal side and 5 on the palatal side of the tooth. The total energy density at each application was 10 J with an interappointment gap of 3 weeks. The retraction was carried using a constant force of 150 gm. A digital vernier caliper measurement was used to measure the distance between the contact points of the maxillary canine and second premolar on 1st and 84th day. RESULTS: The rate of orthodontic tooth movement was faster on the experimental side, and the difference between the two sides was statistically significant (P < 0.014). INTERPRETATION AND CONCLUSION: It was concluded that biostimulation carried out using an 810 nm diode laser is capable of increasing the rate of extraction space closure. Hence, it is capable of increasing the rate of orthodontic tooth movement.

A Comparative Evaluation of Interleukin 1 Beta and Prostaglandin E2 with and without Low-level Laser Therapy during En masse Retraction.

BACKGROUND AND OBJECTIVES: Orthodontic forces are known to produce mechanical damage and inflammatory mediators such as prostaglandins (PGs) and interleukin (IL)-1, in the periodontium and dental pulp. Low-level laser therapy (LLLT) is a stimulator of the on-going biological process in tissue and found to be effective in modulating cell activity, which is involved in orthodontic tooth movement. Here, a humble effort has been made to study two such cytokines, namely IL-1 ß and PG E2 (PGE2) which are partially responsible for bone turnover. The purpose of this study was to compare the changes occurring in the values of IL-1 ß and PGE2 in the gingival crevicular fluid (GCF) during en masse retraction with and without LLLT. METHODOLOGY: GCF was collected using micropipettes from the distal ends of upper canines. The experimental side was exposed to biostimulation using 810 nm gallium-aluminum-arsenide diode laser and the contralateral side taken as control. A total of 10 irradiations for 10 s per site were given, five on the buccal side and five on the palatal side, to cover the entire periodontal fibers and the alveolar process around the tooth. After 7 days and 21 days of retraction, GCF sample was collected. Quantitative analysis of IL-1 ß and PGE2 in the GCF samples was assessed using a commercially available Raybiotech® IL-1 ß and Human PGE2. RESULTS: (1) IL-1 ß and PGE2 levels showed significant results from baseline to 21 days after LLLT irradiation. (2) LLLT-assisted retraction was significantly faster than conventional retraction. INTERPRETATION AND CONCLUSION: It was concluded from the study that IL-1 ß and PGE2 levels peaked after LLLT. The difference in the levels of both cytokines was statistically significant.

Nanostructured Hyaluronic Acid-based Materials for the Delivery of siRNA.

BACKGROUND: The search for the effective treatment strategies to combat a disease that is characterized by abnormal cell growth and known as cancer is still required to reach its destiny. To address the problem, recently several gene therapies based on novel RNA interference (RNAi) have been proposed such as siRNA, micro RNA, shRNA, etc. out of which, siRNAs (silencing RNA) promises to show significant progress in pharmacotherapy, including considerable expansion of the druggable target space and the possibility of treating cancer. METHODS: This review aims to uncover the hyaluronic acid (HA) and HA-hybridized nanoplatforms for siRNA delivery systems with a particular focus on the discussion of available reports while addressing the future potential of HA-based treatment strategies. RESULTS: HA modified siRNA delivery, as promised, provided better targeting potential in many types of cancers. In addition, it was able to modify the release of siRNA as well. Toxicity of HA is well mentioned however, the loophole is yet to be filled by exploring various remedies for overcoming toxicity. CONCLUSION: To overcome the problems associated with these emerging genetic tools, investigators have employed glycosaminoglycan HA-based biopolymers. This biopolymer offers a variety of properties such as biodegradability, biocompatibility, aqueous solubility, viscoelasticity, and non-immunogenicity.

Altered cellular distribution and subcellular sorting of gamma-tubulin in diffuse astrocytic gliomas and human glioblastoma cell lines.

Centrosome amplification is a pivotal mechanism underlying tumorigenesis but its role in gliomas is underinvestigated. The present study specifically examines the expression and distribution of the centrosome-associated cytoskeletal protein gamma-tubulin in 56 primary diffuse astrocytic gliomas (grades II-IV) and in 4 human glioblastoma cell lines (U87MG, U118MG, U138MG, and T98G). Monoclonal anti-peptide antibodies recognizing epitopes in C-terminal or N-terminal domains of the gamma-tubulin molecule were used in immunohistochemical, immunofluorescence, and immunoblotting studies. In tumors in adults (n = 46), varying degrees of localization were detected in all tumor grades, but immunoreactivity was significantly increased in high-grade anaplastic astrocytomas and glioblastomas multiforme as compared to low-grade diffuse astrocytomas (p = 0.0001). A similar trend was noted in diffuse gliomas in children but the sample of cases was too small as to be statistically meaningful. Two overlapping patterns of ectopic cellular localization were identified in both primary tumors and glioblastoma cell lines: A punctate pattern, in which gamma-tubulin was partially co-distributed with pericentrin in the pericentriolar region, and a diffuse pattern, independent of pericentrin staining, denoting a soluble pool of gamma-tubulin. Cellular gamma-tubulin was detected in both soluble and insoluble (nocodazole-resistant) fractions of glioblastoma cells. Divergent localizations of gamma-tubulin and pericentrin suggest a differential distribution of these 2 centrosome-associated proteins in glioblastoma cell lines. Our results indicate that overexpression and ectopic cellular distribution of gamma-tubulin in astrocytic gliomas may be significant in the context of centrosome protein amplification and may be linked to tumor progression and anaplastic potential.

Combination therapy with local radiofrequency ablation and systemic vaccine enhances antitumor immunity and mediates local and distal tumor regression.

PURPOSE: Radiofrequency ablation (RFA) is a minimally invasive energy delivery technique increasingly used for focal therapy to eradicate localized disease. RFA-induced tumor-cell necrosis generates an immunogenic source of tumor antigens known to induce antitumor immune responses. However, RFA-induced antitumor immunity is insufficient to control metastatic progression. We sought to characterize (a) the role of RFA dose on immunogenic modulation of tumor and generation of immune responses and (b) the potential synergy between vaccine immunotherapy and RFA aimed at local tumor control and decreased systemic progression. EXPERIMENTAL DESIGN: Murine colon carcinoma cells expressing the tumor-associated (TAA) carcinoembryonic antigen (CEA) (MC38-CEA(+)) were studied to examine the effect of sublethal hyperthermia in vitro on the cells' phenotype and sensitivity to CTL-mediated killing. The effect of RFA dose was investigated in vivo impacting (a) the phenotype and growth of MC38-CEA(+) tumors and (b) the induction of tumor-specific immune responses. Finally, the molecular signature was evaluated as well as the potential synergy between RFA and poxviral vaccines expressing CEA and a TRIad of COstimulatory Molecules (CEA/TRICOM). RESULTS: In vitro, sublethal hyperthermia of MC38-CEA(+) cells (a) increased cell-surface expression of CEA, Fas, and MHC class I molecules and (b) rendered tumor cells more susceptible to CTL-mediated lysis. In vivo, RFA induced (a) immunogenic modulation on the surface of tumor cells and (b) increased T-cell responses to CEA and additional TAAs. Combination therapy with RFA and vaccine in CEA-transgenic mice induced a synergistic increase in CD4(+) T-cell immune responses to CEA and eradicated both primary CEA(+) and distal CEA(-) s.c. tumors. Sequential administration of low-dose and high-dose RFA with vaccine decreased tumor recurrence compared to RFA alone. These studies suggest a potential clinical benefit in combining RFA with vaccine in cancer patients, and augment support for this novel translational paradigm.

Synthesis and in vitro evaluation of cyclic NGR peptide targeted thermally sensitive liposome.

The Asn-Gly-Arg (NGR) motif in both cyclic and linear form has previously been shown to specifically bind to CD13/aminopeptidase N that is selectively overexpressed in tumor vasculature and some tumor cells. However, previous versions of cyclic NGR used a liable disulfide bridge between cysteine residues that may be problematic for liposome targeting due to disulfide bond formation between adjacent peptides on the liposomal surface. In this study, we report the design, synthesis, and characterization of a novel cyclic NGR-containing peptide, cKNGRE, which does not contain a disulfide bridge. cKNGRE was synthesized in good yield and purity and attached to the fluorescent reporter Oregon Green (cKNGRE-OG) and lysolipid-containing temperature sensitive liposomes (LTSLs). The identity of cKNGRE was verified with NMR and mass spectral techniques. In vitro fluorescence microscopy evaluation of cKNGRE-OG demonstrated binding and active uptake by CD13(+) cancer cells and minimal binding to CD13(-) cancer cells. The cKNGRE-OG ligand displayed 3.6-fold greater affinity for CD13(+) cancer cells than a linear NGR-containing peptide. Affinity for CD13(+) cancer cells was similarly improved 10-fold for both the cyclic and linear NGR when presented in a multivalent fashion on the surface of an LTSL. cKNGRE-targeted LTSLs rapidly released (>75% in <4s) doxorubicin at 41.3 degrees C with minimal release at 37 degrees C. These results demonstrate the ability to synthesize a cKNGRE-targeted temperature sensitive liposome that lacks a disulfide bridge and has sufficient binding affinity for biological applications.

Class III beta-tubulin and gamma-tubulin are co-expressed and form complexes in human glioblastoma cells.

We have previously shown that the neuronal-associated class III beta-tubulin isotype and the centrosome-associated gamma-tubulin are aberrantly expressed in astrocytic gliomas (Cell Motil Cytoskeleton 2003, 55:77-96; J Neuropathol Exp Neurol 2006, 65:455-467). Here we determined the expression, distribution and interaction of betaIII-tubulin and gamma-tubulin in diffuse-type astrocytic gliomas (grades II-IV) (n = 17) and the human glioblastoma cell line T98G. By immunohistochemistry and immunofluorescence microscopy, betaIII-tubulin and gamma-tubulin were co-distributed in anaplastic astrocytomas and glioblastomas and to a lesser extent, in low-grade diffuse astrocytomas (P < 0.05). In T98G glioblastoma cells betaIII-tubulin was associated with microtubules whereas gamma-tubulin exhibited striking diffuse cytoplasmic staining in addition to its expectant centrosome-associated pericentriolar distribution. Treatment with different anti-microtubule drugs revealed that betaIII-tubulin was not associated with insoluble gamma-tubulin aggregates. On the other hand, immunoprecipitation experiments unveiled that both tubulins formed complexes in soluble cytoplasmic pools, where substantial amounts of these proteins were located. We suggest that aberrant expression and interactions of betaIII-tubulin and gamma-tubulin may be linked to malignant changes in glial cells.

Glioneuronal phenotype in a diencephalic pilomyxoid astrocytoma.

We report the presence of divergent populations of cells in a hypothalamic/chiasmatic pilomyxoid astrocytoma of an 11-month-old male, exhibiting differential immunohistochemical localizations for glial fibrillary acidic protein (GFAP) and synaptophysin. The tumor cells were negative for Neu-N and neurofilament protein. Ultrastructurally, the tumor comprised 2 cell types, one with features attributable to a neuronal phenotype alongside cells exhibiting an overt astroglial phenotype. This composite organization was confirmed by confocal microscopy, which revealed 2 distinct, albeit tightly interwoven, populations of GFAP and synaptophysin-labeled tumor cells. Our results indicate that a subset of the so-called pilomyxoid astrocytomas of the hypothalamic/chiasmatic region may represent phenotypically mixed glioneuronal neoplasms distinct from the pilocytic astrocytomas.