Anil K. Bansal

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‐α 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.

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.

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.

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.

Plasma fibronectin stabilizes Borrelia burgdorferi–endothelial interactions under vascular shear stress by a catch-bond mechanism

Significance Spread of bacteria via the bloodstream to vital organs causes most mortality due to bacterial infection. To exit the bloodstream and enter these organs, bacteria must be able to resist the forces generated by flowing blood so that they can adhere to the endothelial cells lining blood vessels without being washed away. This process is not yet understood for most disease-causing bacteria. Here, we show that the Lyme disease pathogen Borrelia burgdorferi exploits an abundant constituent of blood, plasma fibronectin, to form endothelial interactions that become stronger as forces due to blood-flow increase. The ability to recruit this highly conserved molecule may also be important for the vascular interaction mechanisms of other pathogens. Bacterial dissemination via the cardiovascular system is the most common cause of infection mortality. A key step in dissemination is bacterial interaction with endothelia lining blood vessels, which is physically challenging because of the shear stress generated by blood flow. Association of host cells such as leukocytes and platelets with endothelia under vascular shear stress requires mechanically specialized interaction mechanisms, including force-strengthened catch bonds. However, the biomechanical mechanisms supporting vascular interactions of most bacterial pathogens are undefined. Fibronectin (Fn), a ubiquitous host molecule targeted by many pathogens, promotes vascular interactions of the Lyme disease spirochete Borrelia burgdorferi. Here, we investigated how B. burgdorferi exploits Fn to interact with endothelia under physiological shear stress, using recently developed live cell imaging and particle-tracking methods for studying bacterial–endothelial interaction biomechanics. We found that B. burgdorferi does not primarily target insoluble matrix Fn deposited on endothelial surfaces but, instead, recruits and induces polymerization of soluble plasma Fn (pFn), an abundant protein in blood plasma that is normally soluble and nonadhesive. Under physiological shear stress, caps of polymerized pFn at bacterial poles formed part of mechanically loaded adhesion complexes, and pFn strengthened and stabilized interactions by a catch-bond mechanism. These results show that B. burgdorferi can transform a ubiquitous but normally nonadhesive blood constituent to increase the efficiency, strength, and stability of bacterial interactions with vascular surfaces. Similar mechanisms may promote dissemination of other Fn-binding pathogens.

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.

Infection with the Lyme disease pathogen suppresses innate immunity in mice with diet-induced obesity

Obesity is a major global public health concern. Immune responses implicated in obesity also control certain infections. We investigated the effects of high‐fat diet‐induced obesity (DIO) on infection with the Lyme disease bacterium Borrelia burgdorferi in mice. DIO was associated with systemic suppression of neutrophil‐ and macrophage‐based innate immune responses. These included bacterial uptake and cytokine production, and systemic, progressive impairment of bacterial clearance, and increased carditis severity. B. burgdorferi‐infected mice fed normal diet also gained weight at the same rate as uninfected mice fed high‐fat diet, toll‐like receptor 4 deficiency rescued bacterial clearance defects, which greater in female than male mice, and killing of an unrelated bacterium (Escherichia coli) by bone marrow‐derived macrophages from obese, B. burgdorferi‐infected mice was also affected. Importantly, innate immune suppression increased with infection duration and depended on cooperative and synergistic interactions between DIO and B. burgdorferi infection. Thus, obesity and B. burgdorferi infection cooperatively and progressively suppressed innate immunity in mice.

The Lyme disease pathogen Borrelia burgdorferi infects murine bone and induces trabecular bone loss

ABSTRACT Lyme disease is caused by members of the Borrelia burgdorferisensu lato species complex. Arthritis is a well-known late-stage pathology of Lyme disease, but the effects of B. burgdorferi infection on bone at sites other than articular surfaces are largely unknown. In this study, we investigated whether B. burgdorferi infection affects bone health in mice. In mice inoculated with B. burgdorferi or vehicle (mock infection), we measured the presence of B. burgdorferi DNA in bones, bone mineral density (BMD), bone formation rates, biomechanical properties, cellular composition, and two- and three-dimensional features of bone microarchitecture. B. burgdorferi DNA was detected in bone. In the long bones, increasing B. burgdorferi DNA copy number correlated with reductions in areal and trabecular volumetric BMDs. Trabecular regions of femora exhibited significant, copy number-correlated microarchitectural disruption, but BMD, microarchitectural, and biomechanical properties of cortical bone were not affected. Bone loss in tibiae was not due to increased osteoclast numbers or bone-resorbing surface area, but it was associated with reduced osteoblast numbers, implying that bone loss in long bones was due to impaired bone building. Osteoid-producing and mineralization activities of existing osteoblasts were unaffected by infection. Therefore, deterioration of trabecular bone was not dependent on inhibition of osteoblast function but was more likely caused by blockade of osteoblastogenesis, reduced osteoblast survival, and/or induction of osteoblast death. Together, these data represent the first evidence that B. burgdorferi infection induces bone loss in mice and suggest that this phenotype results from inhibition of bone building rather than increased bone resorption.

Vitamin E-mediated protection on methomyl-induced alterations in rat liver

The present study was carried out to observe the possible beneficial effects of Vitamin E, a natural antioxidant on methomyl-induced biochemical and histological alterations in rat liver. To carry out the investigations, animals were segregated in four different groups. Animals in Group I served as normal controls. Animals in Group II were given single methomyl dose orally in water (9 mg kg−1 b.wt). Animals in Group III were injected intraperitoneally with Vitamin E (50 mg kg−1 b.wt) for 1 week on alternate days. Animals in Group IV were administered Vitamin E 1 week before subjecting them to methomyl treatment. Animals in all the groups were sacrificed 24 h after the end of treatments. Different biochemical estimations were carried out, which included estimation of aspartate aminotransaminase (AST), alanine aminotransaminase (ALT), alkaline phosphatase (ALP) and acetylcholinesterase (AChE). Further, to examine the oxidative damage lipid peroxidation (LPO) and glutathione (GSH) levels as well as antioxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GSHPx), and glutathione-6-phosphate dehydrogenase were estimated in liver samples. AchE activity was inhibited significantly both in serum and liver following methomyl treatment. Administration of methomyl caused a significant increase in serum AST, ALT and ALP which indicated hepatic damage. LPO was found to be significantly increased, whereas GSH levels were decreased in the liver of methomyl-treated animals. The activities of SOD and catalase were significantly decreased whereas GST and GSHPx activities were found to be elevated significantly following methomyl treatment. No significant change in the enzyme activity of GR and glutathione-6-phosphatase dehydrogenase was observed after methomyl treatment. Vitamin E supplementation was able to attenuate appreciably the methomyl-induced changes in LPO levels along with SOD and GST activities. Histopathological studies following methomyl treatment revealed that hepatocytes, were not very well delineated and nuclei showed degenerative changes. Whereas, following Vitamin E supplementation in combined treatment group nuclei showing degenerative changes become less in number. The study, therefore, concludes that Vitamin E has a potential in mitigating most of the adverse effects induced by methomyl acute toxicity.