Hyperglycemia Impairs Neutrophil-Mediated Bacterial Clearance in Mice Infected with the Lyme Disease Pathogen.

Insulin-insufficient type 1 diabetes is associated with attenuated bactericidal function of neutrophils, which are key mediators of innate immune responses to microbes as well as pathological inflammatory processes. Neutrophils are central to immune responses to the Lyme pathogen Borrelia burgdorferi. The effect of hyperglycemia on host susceptibility to and outcomes of B. burgdorferi infection has not been examined. The present study investigated the impact of sustained obesity-independent hyperglycemia in mice on bacterial clearance, inflammatory pathology and neutrophil responses to B. burgdorferi. Hyperglycemia was associated with reduced arthritis incidence but more widespread tissue colonization and reduced clearance of bacterial DNA in multiple tissues including brain, heart, liver, lung and knee joint. B. burgdorferi uptake and killing were impaired in neutrophils isolated from hyperglycemic mice. Thus, attenuated neutrophil function in insulin-insufficient hyperglycemia was associated with reduced B. burgdorferi clearance in target organs. These data suggest that investigating the effects of comorbid conditions such as diabetes on outcomes of B. burgdorferi infections in humans may be warranted.

A patient-based dosimetric study of intracavitary and interstitial brachytherapy in advanced stage carcinoma of the cervix.

Intracavitary brachytherapy (ICBT) and interstitial brachytherapy (IB) techniques are commonly practiced for treating carcinoma of the cervix, either alone or in combination with external beam radiotherapy. Both these brachytherapy techniques have their own advantages and limitations in terms of tumor coverage and normal tissue sparing. Limited studies have been reported comparing the dosimetric features of these two techniques, especially from a single institution. We carried out a prospective clinical dosimetric comparison between ICBT and IB for patients treated at one center to bring out the inherent dosimetric features of these to two techniques. The study was carried out on 26 patients treated with ICBT and 55 with IB using CT-based planning. Of the 55 patients treated with IB, 27 included tandem source loading (IBT) and 28 without the tandem loading (IBWT). The high-dose volumes covered by 200% and 180% isodose surfaces were considerably larger in ICBT as compared to IB, whereas the treated volume was larger in IB as compared to ICBT. The bladder and rectal doses were the highest in ICBT and IBWT, respectively. The larger treated volume in IB as compared to ICBT was mainly because patients with larger tumor volumes were generally considered for IB. The results also indicated that in interstitial brachytherapy, better rectal sparing was achieved by including the tandem for treatment delivery.

Fractionated stereotactic radiosurgery with volumetric modulated arc therapy (Rapid Arc) for reradiation in recurrent high grade gliomas.

BACKGROUND: To evaluate 'Rapid Arc (RA)' technique for delivering fractionated stereotactic radiosurgery (FSRS) in patients with recurrent high grade gliomas (HGGs) for minimizing the dose to previously radiated high dose brain volume. MATERIALS AND METHODS: Between April 2010 and February 2011, 16 consecutive patients with recurrent HGGs and previously treated with intensity modulated radiation therapy (IMRT) and Temozolamide received FSRS. The median time between IMRT and FSRS was 10.72 months. FSRS to a dose of 30 Gy in a median of 5 fractions was delivered to the recurrent tumor (gross tumor volume [GTV]). Brain volume around the GTV and previously treated to a mean dose >50 Gy was delineated as "Avoidance Volume (AV)." Patients were planned with both RA and Dynamic Conformal Arc (DCA) to achieve minimum dose to AV. Dose received by GTV, AV, rest of the normal brain (brain minus PTV) and conformity index (CI) and heterogenecity index (HI) were compared by the two techniques. RESULTS: At a median follow up of 7.33 months, median progression free and overall survival was 6.4 and 9.3 months, respectively. Mean dose to AV was significantly lower with RA as compared with DCA (10.8 Gy vs. 15.5 Gy, P - 0.0001) with no significant difference in the dose delivered to GTV. No patient developed radiation necrosis. CONCLUSION: As compared with DCA, RA delivered significantly less dose to previously radiated high dose brain volume. It may contribute to minimizing the risk of radionecrosis with stereotactic radiosurgery (SRS) in patients with recurrent HGG.

A phantom study on bladder and rectum dose measurements in brachytherapy of cervix cancer using FBX aqueous chemical dosimeter.

The ferrous sulphate-benzoic acid-xylenol orange (FBX) chemical dosimeter, due to its aqueous form can measure average volume doses and hence may overcome the limitations of point dosimetry. The present study was undertaken to validate the use of FBX dosimeter for rectum and bladder dose measurement during intracavitary brachytherapy (ICBT) and transperineal interstitial brachytherapy (TIB). We filled cylindrical polypropylene tubes (PT) and Foley balloons (FB) with FBX solution and used them as substitutes for rectum and bladder dose measurements respectively. A water phantom was fabricated with provision to place the Fletcher-type ICBT and MUPIT template applicators, and FBX filled PT and FB within the phantom. The phantom was then CT scanned for treatment planning and subsequent irradiation. Our results show that the average difference between DVH derived dose value and FBX measured dose is 3.5% (PT) and 13.7% (FB) for ICBT, and 9% (PT) and 9.9% (FB) for TIB. We believe that the FBX system should be able to provide accuracy and precision sufficient for routine quality assurance purposes. The advantage of the FBX system is its water equivalent composition, average volume dose measuring capability, and energy and temperature independent response as compared to TLD or semiconductor dosimeters. However, detailed studies will be needed with regards to its safety before actual in-vivo dose measurements are possible with the FBX dosimeter.

Celiac G+ antibody assay for the detection of autoantibodies in celiac disease.

Celiac disease (CD) affects approximately 1% of the population and may present with varied symptomatic as well as asymptomatic clinical manifestations. Simple methods of detecting CD such as serum antibody tests have helped in the early identification of the disease thus preventing serious complications of the disorder. Our objective is to develop specific and sensitive immunoassays that are reliable in the detection of CD. To this end, immunoassays were developed for the detection of IgG and IgA antibodies to gliadin using synthetic peptides. Over 200 serum samples were included in the study from individuals with CD submitted for endomysial (EMA) and tissue transglutaminase (tTG) antibody tests as well as from disease controls and healthy normals. To examine the reliability of the Celiac G+ antibody test in comparison with EMA, a test with higher sensitivity and specificity, samples with low and high EMA titers were included in the study. Comparative evaluations of the Celiac G+ antibody assay were made with EMA and another commercially available gliadin peptide assay along with tTG antibody assays. The data show that as the EMA levels increased the sensitivity of detection of antibodies to synthetic peptides on both systems increased, reaching 100% at EMA titers greater than 160. The diagnostic performance of the newly developed Celiac G+ synthetic gliadin peptide assay is significantly superior in comparison with another available gliadin peptide immunoassay. Overall, the diagnostic performance of the Celiac G+ assay for IgA and IgG reached a sensitivity of 80% and 90% respectively in comparison with EMA. Similar comparison of the EMA positivity to the other available synthetic peptide immunoassay yielded sensitivities of 59% (IgA) and 75% (IgG). The specificity of the Celiac G+ antibody assay for IgA and IgG was 90-95% as compared to the other similar assay with specificity of 88-90%. In conclusion, the performance of the recently developed Celiac G+ ELISA is superior in both its sensitivity and specificity in comparison with other available synthetic gliadin peptide immunoassays. Furthermore, the IgG Celiac G+ antibody test and IgA tTG antibody test used in combination is an excellent screening algorithm for suspected cases of celiac disease.

Characterisation of the constitutive over-expression of AJ18 in a novel rat stromal bone marrow cell line (D8-SBMC).

OBJECTIVE: The elucidation of the molecular pathways involved in osteoblast proliferation and differentiation has been greatly enhanced by the availability of cell culture model systems. However, many of the current bone cell culture systems suffer from disadvantages such as the inability to generate mineralised bone-like nodules, a transformed genetic background, cell heterogeneity, and a relatively long time frame from cell seeding to mineralisation, often in the order of several weeks. Here we describe the establishment and characterisation of a novel bone cell line named D8-SBMC. As a first demonstration of their potential value, D8-SBMC was utilised to further support a role for AJ18 during osteogenesis. DESIGN: D8-SBMC was established from a single cell suspension of the previously characterised long term rat stromal bone marrow cells [Kotev-Emeth S, Pitaru S, Pri-Chen S, Savion N. Establishment of a rat long-term culture expressing the osteogenic phenotype: dependence on dexamethasone and FGF-2. Connect Tissue Res 2002;43(4):606-12; Pitaru S, Kotev-Emeth S, Noff D, Kaffuler S, Savion N. Effect of basic fibroblast growth factor on the growth and differentiation of adult stromal bone marrow cells: enhanced development of mineralized bone-like tissue in culture. J Bone Miner Res 1993;8(8):919-29]. AJ18 was constitutively and stably over-expressed in D8-SBMC and analysed. RESULTS: D8-SBMC possesses the ability to form robust mineralised bone-like nodules within 8 days proceeding cell confluency. Interestingly, a cement line-like matrix is also generated between the culture dish and a basal monolayer of cells. Constitutive and stable over-expression of AJ18 resulted in an increase in cell proliferation and mineralisation. Expression of bone marker genes, such as bone sialoprotein, osteopontin, osteocalcin, collage type 1, and osteonectin, was up-regulated by AJ18 over-expression. CONCLUSION: A novel bone cell line, D8-SBMC, was established and characterised. D8-SBMC may be a valuable model system for biomineralisation studies. D8-SBMC was utilised to further understand the role of AJ18 in cell proliferation and differentiation during osteogenesis.

FBX aqueous chemical dosimeter for measurement of virtual wedge profiles.

We investigated the ferrous sulfate-benzoic acid-xylenol orange (FBX) aqueous chemical dosimeter for measurement of virtual (dynamic) wedge profiles on a linear accelerator. The layout for irradiation of the FBX-filled tubes mimicked a conventional linear detector array geometry. A comparison of the resulting measurements with film-measured profiles showed that, in the main beam region, the difference between the FBX system and the film system was within +/-2% and that, in the penumbra region, the difference varied from +/-1 mm to +/-2.5 mm in terms of positional equivalence, depending on the size of the dosimeter tubes. We thus believe that the energy-independent FBX dosimetry system can measure virtual wedge profiles with reasonable accuracy at reasonable cost. However, efficiency improvement is required before this dosimetry system can be accepted into routine practice.

Role of vitamin E in mitigating methomyl induced acute toxicity in blood of male Wistar rats.

The present study was designed to evaluate the protective potential of vitamin E, if any, in attenuating the toxic effects induced by acute methomyl treatment in rats. Male Wistar rats, weighing between 230 and 250 g, received either a single oral dose of 9 mg/kg of methomyl, vitamin E alone injected intraperitoneally on alternate days (4 injections) at 50 mg/kg body for 1 week prior to methomyl treatment, or both methomyl plus vitamin E given in a similar manner. The effects of different treatments were studied on lipid peroxidation (LPO), reduced glutathione (GSH) and antioxidant enzymes, which included superoxide dismutase (SOD), glutathione-s-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GSHPx) and catalase and various hematological parameters, including total leucocytes count (TLC), differential leukocyte count (DLC), hemoglobin, platelets counts, red cell counts, and scanning electron microscopy (SEM). Acute 24-h treatment to rats resulted in a significant increase in the LPO. GSH levels and the activities of catalase, GST, and GSHPx were found to be significantly decreased following methomyl treatment. A significant elevation in the activity of SOD and in TLC was also observed after 24 h of methomyl treatment. Further, a significant increase in the neutrophils and eosinophil counts was also observed. However, lymphocytes showed a significant decrease following methomyl treatment. SEMs showed significant morphological changes following methomyl treatment. Vitamin E pretreatment to methomyl-treated rats effectively normalized the levels of LPO and GSH. Vitamin E could also significantly elevate the activity of catalase, increase platelets counts and TLC, and normalized the activities of SOD and GSHPx. Vitamin E pretreatment improved the morphology of the red blood cells. The study concludes that vitamin E affords protection in methomyl-induced toxicity in the rat.

Role of osteopontin in neutrophil function.

Osteopontin (OPN) is important for the function of fibroblasts, macrophages and lymphocytes during inflammation and wound healing. In recent studies of experimental colitis we demonstrated exacerbated tissue destruction in OPN-null mice, associated with reduced tumour necrosis factor-alpha expression and increased myeloperoxidase activity. The objective of this investigation therefore was to determine the importance of OPN expression in neutrophil function. Although, in contrast to macrophages, neutrophils expressed low levels of OPN with little or no association with the CD44 receptor, intraperitoneal recruitment of neutrophils in OPN-null mice was impaired in response to sodium periodate. The importance of exogenous OPN for neutrophil recruitment was demonstrated by a robust increase in peritoneal infiltration of PMNs in response to injections of native or recombinant OPN. In vitro, OPN(-/-) neutrophils exhibited reduced chemokinesis and chemotaxis towards N-formyl methionyl leucyl phenylalanine (fMLP), reflecting a reduction in migration speed and polarization. Exogenous OPN, which was chemotactic for the neutrophils, rescued the defects in polarization and migration speed of the OPN(-/-) neutrophils. In contrast, the defensive and cytocidal activities of OPN(-/-) neutrophils, measured by assays for phagocytosis, generation of reactive oxygen species, cytokine production and matrix metalloproteinase-9, were not impaired. These studies demonstrate that, while exogenous OPN may be important for the recruitment and migration of neutrophils, expression of OPN by neutrophils is not required for their destructive capabilities.

Impact of different breathing conditions on the dose to surrounding normal structures in tangential field breast radiotherapy.

Cardiac toxicity is an important concern in tangential field breast radiotherapy. In this study, the impact of three different breathing conditions on the dose to surrounding normal structures such as heart, ipsilateral lung, liver and contralateral breast has been assessed. Thirteen patients with early breast cancer who underwent conservative surgery (nine left-sided and four right-sided breast cancer patients) were selected in this study. Spiral CT scans were performed for all the three breathing conditions, viz., deep inspiration breath-hold (DIBH), normal breathing phase (NB) and deep expiration breath-hold (DEBH). Conventional tangential fields were placed on the 3D-CT dataset, and the parameters such as V30 (volume covered by dose >30 Gy) for heart, V20 (volume covered by dose >20 Gy) for ipsilateral lung and V(50) (volume receiving >50% of the prescription dose) for heart and liver were studied. The average reduction in cardiac dose due to DIBH was 64% (range: 26.5-100%) and 74% (range: 37-100%) as compared to NB and DEBH respectively. For right breast cancer, DIBH resulted in excellent liver sparing. Our results indicate that in patients with breast cancer, delivering radiation in deep inspiration breath-hold condition can considerably reduce the dose to the surrounding normal structures, particularly heart and liver.

Protective role of Vitamin E pre-treatment on N-nitrosodiethylamine induced oxidative stress in rat liver.

Nitrosamine compounds are known hepatic carcinogens. In the metabolism of nitrosamines, such as N-nitrosodiethylamine (NDEA), there is evidence of the formation of reactive oxygen species (ROS) resulting in oxidative stress, which may be one of the factors in the etiology of cancer. The formation of ROS may alter the antioxidant system, while the presence of Vitamin E may counteract NDEA induced oxidative stress. This study was planned to determine whether pre-treatment with Vitamin E (40 mg/kg body weight, i.p., twice a week for 4 weeks) to NDEA induced rats provides protection against oxidative stress in liver caused by the carcinogen. A single necrogenic dose of NDEA (200mg/kg body weight) was administered i.p. to the male albino rats with or without Vitamin E pre-treatment and the animals were sacrificed on Days 7, 14 or 21 after the administration of NDEA. The result showed enhanced levels of hepatic lipid peroxidation (LPO) and conjugated dienes of NDEA treated rats as the indices of oxidative stress, however, Vitamin E pre-treated rats administered NDEA showed decreased LPO and conjugated dienes (Day 21). Superoxide dismutase (SOD) activity in liver was not altered significantly in NDEA treated rats with or without Vitamin E pre-treatment. Catalase (CAT) activity was inhibited with NDEA treatment, however, Vitamin E pre-treatment showed recovery in hepatic CAT activity (Days 14 and 21). Total and Se-glutathione peroxidase (GSH-Px) activities and glutathione-S-transferase (GST) activity in liver increased in NDEA treated rats irrespective of Vitamin E pre-treatment. Glutathione reductase (GSH-R) activity as well as total glutathione (GSH) content in liver decreased in NDEA treated animals, both of which were recovered in Vitamin E pre-treated rats administered NDEA. Activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were increased significantly following NDEA treatment to rats with or without Vitamin E pre-treatment. The activities of AST and ALT enzymes were significantly reduced on Days 14 and 21 and ALP activity was reduced on Day 21 in NDEA+Vitamin E treated animals when compared to NDEA treated alone. LDH enzyme activity was normalized on Day 14 in Vitamin E pre-treated animals administered NDEA. However, the AST, ALT and ALP enzyme activities remained high in all treatment groups as compared to control group. Normal control and Vitamin E treated alone rats revealed normal histology of liver. On the other hand, NDEA treated animals showed alterations in normal hepatic histoarchitecture, which comprised of necrosis and vacuolization of the cells. However, the rats treated with Vitamin E+NDEA showed that the liver cells were normal, with very little necrosis (Day 21). This study concludes that the pre-treatment with Vitamin E prior to the administration of NDEA, reduced the degree of oxidative stress, although this vitamin produced only slight changes in the hepatic injury, in a time-dependent manner.

Recruitment of nuclear factor Y to the inverted CCAAT element (ICE) by c-Jun and E1A stimulates basal transcription of the bone sialoprotein gene in osteosarcoma cells.

Bone sialoprotein (BSP), a major protein in the extracellular matrix of bone, is expressed almost exclusively by bone cells and by cancer cells that have a propensity to metastasize to bone. Previous studies have shown that v-src stimulates basal transcription of bsp in osteosarcoma (ROS 17/2.8) cells by targeting the inverted CCAAT element (ICE) in the proximal promoter. To identify possible downstream effectors of Src we studied the effects of the proto-oncogene c-jun, which functions downstream of Src, on basal transcription of bsp using transient transfection assays. Increased expression of endogenous c-Jun induced by the tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate and ectopic expression of c-Jun increased basal transcription of chimeric reporter constructs encompassing the proximal promoter by 1.5-3-fold in ROS 17/2.8 osteosarcoma cells, with more modest effects in a normal bone cell line, RBMC-D8. The effects of c-Jun were abrogated by mutations in the ICE box and by co-expression of dominant negative nuclear factor Y, subunit A (NF-YA). The increase in bsp transcription did not require phosphorylation of c-Jun and was not altered by trichostatin treatment or by ectopic expression of p300/CREB-binding protein (CBP) or mutated forms lacking histone acetyltransferase (HAT) activity. Similarly, ectopic expression of p300/CBP-associated factor (P/CAF), which transduces p300/CBP effects, or of HAT-defective P/CAF did not influence the c-jun effects. Surprisingly, E1A, which competes with P/CAF binding to p300/CBP, also stimulated BSP transcription through NF-Y independently of c-jun, p300/CBP, and P/CAF. Collectively, these studies show that c-Jun and E1A regulate basal transcription of bsp in osteosarcoma cells by recruiting the NF-Y transcriptional complex to the ICE box in a mechanism that is independent of p300/CBP and P/CAF HAT activities.

Analysis of human bone sialoprotein in normal and pathological tissues using a monoclonal antibody (BSP 1.2 mab).

Bone sialoprotein (BSP), a phosphorylated and sulphated glycoprotein that is expressed by mineralized connective tissues is also produced in tumors that metastasize to bone. To facilitate studies of BSP expression in normal and pathological human tissues a monoclonal antibody (BSP 1.2 mab) was raised against human bone BSP. BSP 1.2 mab was shown by ELISA assays to recognize the epitope "DEYSY" (amino acids 279-283) that is conserved in mammalian BSP sequences. However, whereas the antibody recognized recombinant BSPs expressed in bacteria, it did not recognize native forms of rat or pig BSP in which the first tyrosine of the DEYSY peptide sequence appears to be modified. Immunostaining of embryonic human tibiae and calvariae with BSP 1.2 mab showed strong reaction in osteoblasts and osteocytes with relatively weak staining of the bone matrix, suggesting that the BSP 1.2 mab epitope is partially masked in the bone matrix. BSP 1.2 mab also stained osteosarcoma cells and normal trophoblastic cells in the placenta in areas of microcrystalline deposits. Cancer cells in primary breast tumors, lymph nodes, and secondary bone metastases from individual patients were stained strongly by BSP 1.2 mab. Although BSP 1.2 mab also stained breast cancer carcinoma cell lines and SaOS2 osteosarcoma cells, biosynthesis of radiolabelled BSP could not be demonstrated in breast cancer cells. Notably, the staining of BSP in the breast cancer cells was diffuse contrasting the punctate staining, typical of secreted proteins, in SaOS2 cells. These studies, therefore, have identified a unique epitope in human BSP recognized by a monoclonal antibody, BSP 1.2 mab, which can be used for the unequivocal identification of BSP in normal and pathological human tissues.