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Biomedical subjects

G Shankar

Publications and source records attributed to G Shankar.

At least 19 recordsLinked to original sources
PubMed2001

Idiopathic pneumonia syndrome after bone marrow transplantation: the role of pre-transplant radiation conditioning and local cytokine dysregulation in promoting lung inflammation and fibrosis.

Pulmonary complications and graft-vs.-host disease (GVHD) remain severe threats to survival after bone marrow transplantation (BMT). Idiopathic pneumonia syndrome (IPS) accounts for nearly 50% of all the cases of interstitial pneumonitis after BMT. IPS is characterized by an early inflammatory phase followed by chronic inflammation and fibrosis of lung tissue; however, the immunopathogenesis of this disease is not yet clearly understood. This biphasic syndrome has been reported to be associated with pre-transplant radiation conditioning in some studies while others have suggested that GVHD or autoimmune phenomena may be responsible for its development. The early post-BMT phase is characterized by the presence of inflammatory cytokines whose net effect is to promote lymphocyte influx into lungs with minimal fibrosis, that leads to an acute form of graft-vs.-host reaction-mediated pulmonary tissue damage. Gradual changes over time in leucocyte influx and activation lead to dysregulated wound repair mechanisms resulting from the shift in the balance of cytokines that promote fibrosis. Using data from new animal models of IPS and information from studies of human IPS, we hypothesize that cytokine-modulated immunological mechanisms which occur during the acute and chronic phases after bone marrow transplantation lead to the development of the progressive, inflammatory, and fibrotic lung disease typical of idiopathic pneumonia syndrome.

Animals↗
PubMed2000

American Association for Cancer Research: potential target antigens for cancer immunotherapy. April 1-5, 2000, San Francisco, CA, USA.

The 91st annual meeting of the American Association for Cancer Research (AACR) convened this year in San Francisco, USA. Nearly 5600 abstracts were published within ten broad research categories ranging from basic molecular, cellular and pharmaceutical research to cancer epidemiology and prevention. The largest number of poster presentations belonged in the 'Pharmacological and Experimental Therapeutics' section, followed closely by 'Molecular Biology' and 'Cell and Tumour Biology'. Novel tumour antigens and their potential application in cancer immunotherapy were displayed primarily in a section categorised as 'Immunology/Preclinical and Clinical'. This article reports some of the significant tumour antigen-related studies presented at this meeting.

Antigens, Neoplasm↗
PubMed2000

Immune monitoring of cancer patients undergoing experimental immunotherapy.

Advancements in the understanding of cellular immunity within the last decade, along with the characterization of tumor antigens, have led to immunotherapeutic approaches for cancer therapy. This mode of treatment is expected to provide more tumor-specific activity, thereby being less toxic to normal cells than standard modalities. Clinical trials are underway throughout the world to determine whether immunotherapy is a practical and viable alternative to conventional cancer therapies. Unlike radiotherapy and chemotherapy, wherein tumor regression is the standard for determining efficacy of the regimens, immunotherapy has to be evaluated by the examination of several immunological parameters within patients. The purpose of this article is to review the methods currently utilized to evaluate the induction, maintenance, and duration of antitumor immune reactivity in cancer patients undergoing immunotherapy.

Antibodies, Neoplasm↗
PubMed1999

Bacterial lipopolysaccharide induced B cell activation is mediated via a phosphatidylinositol 3-kinase dependent signaling pathway.

Bacterial lipopolysaccharide (LPS) is a potent stimulant of B cells and macrophages. LPS induces B cell proliferation and differentiation into antibody secreting cells. In addition, LPS also stimulates IL-6 secretion in mature B cells and in immature B cell lines such as WEHI-231. Although sufficient literature is available on LPS induced signaling events in monocytes and macrophages, the mechanisms involved in LPS induced B cell activation are not well understood. In this report, it is shown that both LPS mediated B cell proliferation and IL-6 secretion are dependent on phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathways. The B cell specific co-receptor, CD19 is not tyrosine phosphorylated in LPS stimulated B cells. Thus, in contrast to B cell antigen receptor (BCR) signaling, the activation of PI 3-kinase appears not to be related to the recruitment of PI 3-kinase to tyrosine phosphorylated CD19. This is the first demonstration of the importance of PI 3-kinase signaling pathway in LPS mediated B lymphocyte activation.

Androstadienes↗
PubMed1999

Protein-kinase-specific inhibitors block Langerhans' cell migration by inhibiting interleukin-1alpha release.

Previous studies have shown that depletion of Langerhans' cells (LC) from murine epidermis by the superantigen, staphylococcal enterotoxin A (SEA) involves interleukin-1alpha (IL-1alpha) and is inhibitable by agents that block G-protein-associated kinases. The purpose of this study was to determine whether specific kinase inhibitors block LC depletion by inhibiting IL-1alpha release and to ascertain whether LC depletion by SEA involves cell migration. These goals were addressed by measuring the IL-1alpha release within whole or LC-depleted epidermal cell suspensions in the presence of SEA and/or H-7 (an inhibitor of protein kinase C) or H-8 (an inhibitor of G-protein-associated kinases) and by examining the migration of cells with LC markers in SEA-treated skin sections. The results suggest that LC depletion by SEA involves migration and that this migration is blocked by protein kinase inhibitors, at least in part, through inhibition of SEA-induced IL-1alpha release by epidermal cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

Idiopathic pneumonia syndrome after allogeneic bone marrow transplantation in mice. Role of pretransplant radiation conditioning.

Idiopathic pneumonia syndrome (IPS) is a significant clinical problem encountered among patients treated with bone marrow transplantation (BMT). IPS is identified as an inflammatory lung disease characterized by diffuse interstitial pneumonitis and alveolitis leading to interstitial fibrosis in the absence of an identifiable infectious agent. In an earlier study we characterized a murine model of IPS following allogeneic BMT that exhibits several features of human IPS. In this report we show that the lung represents a unique target of post-BMT disease in this model. The kinetics of developing lung disease were found to be markedly different from the kinetics of graft-versus-host disease in other tissues such as liver, colon, ear, skin, and tongue. Mice transplanted by our standard protocol with T-cell-depleted semiallogeneic donor bone marrow plus donor spleen cells in the absence of pretransplant radiation conditioning did not develop lung inflammation or fibrosis characteristic of IPS. Pretransplant radiation conditioning in the absence of BMT also failed to cause IPS, demonstrating an important role for radiation conditioning in the development of BMT-related IPS. The occurrence of lung disease post-BMT was found to be dependent on radiation conditioning in a dose-dependent manner. Finally, thoracic irradiation alone was demonstrated to be sufficient in causing IPS in mice transplanted with bone marrow plus spleen cells, albeit with reduced severity. Based on these findings, we conclude that pretransplant radiation conditioning plays an important role in the development of IPS following allogeneic BMT.

Animals↗
PubMed1998

Idiopathic pneumonia syndrome in mice after allogeneic bone marrow transplantation.

Pulmonary complications are a major clinical problem following allogeneic bone marrow transplantation (BMT), contributing to more than 30% of transplant-related mortalities. Idiopathic pneumonia syndrome is responsible for significant mortality among BMT patients. However, the etiology of injury to the lung parenchyma by this disease syndrome is unknown and it has been difficult to evaluate the cellular and molecular mechanisms underlying IPS in the absence of a suitable animal model. To study post-BMT lung disease during graft-versus-host disease (GVHD), we have developed a murine model that utilizes a semi-allogeneic parental --> F1 transplant strategy to induce a mild form of GVHD. Progressive inflammatory lung disease developed in animals with mild GVHD, as indicated by changes in immune cell distribution and cytokine expression in the lungs of transplanted animals. Histologic analysis of lung tissue from GVHD mice at 3 wk post-BMT showed minor immunopathologic changes compared with control mice. In contrast, lungs of GVHD mice at 12 wk displayed histopathologic hallmarks of interstitial pneumonitis, such as prominent perilumenal mononuclear cell infiltration and areas of alveolar congestion. Flow cytometric analysis of lung interstitial cells of GVHD mice revealed an increase in CD8+ T-cells at week 3, which decreased to normal levels by week 12 post-BMT. Simultaneously, the percentage of CD4+ T-cells increased progressively above normal levels and peaked at week 7 post-BMT. Analysis of cytokine mRNA expression in lung tissue indicated that steady state levels of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, interferon-gamma, and IL-12 were significantly elevated in lungs of GVHD mice at 3 wk post-BMT compared with untreated controls. Mice that were transplanted with allogeneic bone marrow alone (BMT controls) also displayed elevated expression of these cytokines, although only IL-6 was significantly higher than in untreated controls. In contrast, at 12 wk after transplantation only TNF-alpha and IL-12 levels remained elevated in GVHD mice, suggesting prolonged macrophage activation. On the basis of these findings, we conclude that allogeneic bone marrow transplantation in this mouse model causes a progressive interstitial pneumonitis, which is characterized by an acute influx of CD8+ T-cells, followed in the chronic phase by a prominent accumulation of CD4+ T-cells, and is associated with persistent production of cytokines known to activate macrophages.

Animals↗
PubMed1997

Enhanced cytokine detection by a novel cell culture-based ELISA.

Production of some cytokines, such as IL-4 and IL-10, often occurs at low levels and is difficult to detect by standard ELISA techniques. In many cases the level of detection is at or near to the limits of sensitivity of the assay due either to minimal synthesis and/or cytokine consumption. In an effort to enhance the quantitation of weakly detected cytokines we have developed a unique cell culture-capture ELISA. Lymphocytes are incubated in an anti-cytokine antibody coated ELISA plate for the last 6 hours of a 24 hour in vitro activation period. Use of this cell culture capture method consistently enhanced detection of several T cell cytokines compared to conventional ELISA techniques. Moreover, this technique was found to enhance detection without altering the rate of cytokine secretion which occurred prior to the cell culture capture period. Thus, the cell culture capture ELISA may be useful for detection of a variety of cytokines which are produced at low levels and have traditionally been difficult to quantify.

Animals↗
PubMed1996

Superantigen-induced Langerhans cell depletion is mediated by epidermal cell-derived IL-1 alpha and TNF alpha.

The purpose of this study was to examine the role of cytokines in staphylococcal enterotoxin-A (SEA)-induced epidermal Langerhans cell (LS) depletion. This was accomplished by analyzing the effect of SEA on cytokine secretion by cultured epidermal cell populations by means of ELISA and by assessing the capacity of cytokines and cytokine-specific antibodies to affect epidermal LC density (as measured by immunoperoxidase staining of epidermal cells bearing surface Ia). The results of this study indicate that SEA induces the secretion of IL-1 alpha and TNF alpha by epidermal cells, that these cytokines induce LC depletion from epidermis, and that antibodies specific for these agents inhibit the depletion of LC by SEA. Taken together, these findings suggest that IL-1 alpha and TNF alpha may play essential roles in SEA-mediated depletion of LC from murine epidermis.

Animals↗
PubMed1996

Beta 1 integrins and osteoclast function: involvement in collagen recognition and bone resorption.

The extracellular matrix of bone is composed mainly of type I collagen. In this report we studied the role and collagen-binding properties of osteoclast integrins (alpha v, alpha 2, beta 1, and beta 3). Cell adhesion assays with rat osteoclasts and affinity chromatography/SDS-PAGE analysis with purified human osteoclast membranes demonstrated adhesion of osteoclasts to native type I collagen in a divalent cation and Arg-Gly-Asp (RGD)-dependent way via alpha 2 beta 1 integrin, whereas osteoclast adhesion to denatured collagen predominantly involved alpha v beta 3. In receptor-binding assays, the involvement of human recombinant alpha v beta 3 in adhesion to denatured collagen was confirmed. Additionally, osteoclasts adhered to type I collagen fibers and to monomeric types II-V collagen with characteristics similar to those on native monomeric type I collagen. Osteoclastic bone resorption in vitro was inhibited (> 40%) in the presence of alpha 2 and beta 1 antibodies. Using scanning laser confocal microscopy, alpha v beta 3, alpha 2, and beta 1 integrin were detected within podosomes in nonresorbing osteoclasts and in the ruffled border area and basolateral membrane in resorbing osteoclasts, but not in the sealing zone of resorbing osteoclasts. These results demonstrate that alpha 2 beta 1, in addition to alpha v beta 3, has an important role in osteoclast function and acts as a receptor for native, but not denatured, collagen.

Amino Acid Sequence↗
PubMed1995

Structural determinants of calcium signaling by RGD peptides in rat osteoclasts: integrin-dependent and -independent actions.

Extensive characterization of the vitronectin receptor (VNR), a member of the integrin group of cell adhesion molecules, which is abundantly expressed in osteoclasts, has revealed a role for this receptor in osteoclast adhesion as well as bone resorption. Earlier evidence from our laboratory suggests that VNR is also capable of transducing intracellular signals following receptor ligand interaction, although this function is poorly understood. Thus, addition of peptides containing the minimal tripeptide Arg-Gly-Asp (RGD) integrin recognition sequence elicits transient increases in intracellular free calcium ions, with maximal responses seen with a bone sialoprotein peptide, BSP-IIA. In the present study we have attempted to determine some of the structural requirements for calcium signaling in osteoclasts using derivatives of the peptide PRGDN/T sequence found in bone sialoprotein. While some peptides, such as the parent sequence PRGDN, can induce both signaling and retractile events, it was found that minor structural modifications yielded peptides such as PRADN which elicited a transient increase in intracellular free calcium ions without promoting a reduction in osteoclast spread area (retraction). Conversely, certain other modifications resulted in peptides, such as PrGDN and benzoyl-RGDN, which effect osteoclast retraction, while having minimal Ca2+ signaling capabilities. Osteoclast adhesion, and hence retraction, are known to be RGD-dependent and integrin-dependent events. However, intracellular Ca2+ signaling is RGD-independent and, based on lack of inhibition by an anti-beta 3 integrin antibody F11 and echistatin, very likely integrin-independent. These data suggest that signaling is not always via VNR and as yet unknown receptors on the osteoclast membrane play a role in osteoclast signaling and hence function.

Amino Acid Sequence↗
PubMed1994

Langerhans' cell depletion by staphylococcal superantigens.

Superantigens were examined for effects on the distribution of Langerhans' cells (LC) in mouse skin. This was accomplished by analysing the expression of LC-specific markers, ATPase and IA among the epidermal portion of cultured sections of mouse skin following treatment with staphylococcal enterotoxins. In this study, treatment of skin sections with staphylococcal enterotoxin A or exfoliative toxin but not toxic shock syndrome toxin led to significant depletion of LC. This depletion was inhibited by agents which specifically block the action of GTP binding proteins or their associated kinases (cholera and pertussis toxins and H-8) as well as those which block protein or RNA synthesis. Therefore, signals which lead to LC depletion in response to staphylococcal enterotoxins appear to involve a cholera and pertussis toxin-sensitive GTP-binding protein and protein synthesis. These requirements are identical to those observed previously for LC depletion following exposure of skin to ultraviolet radiation.

Animals↗
PubMed1993

Integrin receptor-mediated mobilisation of intranuclear calcium in rat osteoclasts.

Cell-matrix interactions have been shown to play an important role in regulating cell function and behaviour. In bone, where calcified matrix formation and resorption events are required to be in dynamic equilibrium, regulation of adhesive interactions between bone cells and their matrix is critical. The present study focuses on the osteoclast, the bone resorbing cell, as well as integrins, which are cell surface adhesion receptors that mediate osteoclast attachment to bone matrix. In osteoclasts, the most abundant integrin receptor is the vitronectin receptor (VNR, alpha v beta 3). The objective of the study was to investigate changes in intracellular calcium, a regulator of osteoclast function, following addition of peptides that bind integrins, in particular the alpha v beta 3 form of the vitronectin receptor (VNR), which is highly expressed in osteoclasts. The study demonstrated a unique spatial localisation of the calcium signal in response to cell membrane receptor occupancy by integrin ligands in rat osteoclasts. Addition of peptides with the Arg-Gly-Asp (RGD) sequence such as BSP-IIA, GRGDSP and GRGDS to rat osteoclasts evoked an immediate increase in free calcium ion concentration [Ca2+]i, localised to the nuclei and to the thin cytoplasmic skirt. These responses were inhibited by F11, a monoclonal antibody to the rat integrin beta 3 chain, as well as echistatin, a snake venom shown to colocalise with the alpha v chain in osteoclasts, suggesting that the calcium signal is mediated by the alpha v beta 3 form of VNR.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Interactions of tumor necrosis factor with local and systemic factors in fetal rat limb bones.

In many tissues the actions of tumor necrosis factor-alpha (TNF) are indirectly mediated through the production of autacoids or other cytokines. To determine the role that these factors might have in the action of TNF on bone resorption, we examined the effects of several selective inhibitors on TNF-stimulated resorption. The cyclooxygenase inhibitor indomethacin did not prevent TNF-stimulated resorption in fetal rat limb bones. Stimulation of resorption by TNF was also unaffected by the platelet activating factor antagonist WEB 2086. A 17.5 kD interleukin receptor antagonist protein, at concentrations that completely blocked the bone-resorbing actions of maximally effective concentrations of interleukin (IL)-1 beta, failed to affect the stimulatory actions of TNF. TNF-stimulated resorption was inhibited by both interferon-gamma and dexamethasone. Dexamethasone inhibited TNF-stimulated resorption more effectively than it inhibited parathyroid hormone (PTH)-stimulated resorption. When bones were treated simultaneously with low concentrations of TNF and PTH, potentiation of the bone-resorbing effects was elicited. These results suggest that TNF stimulates resorption through a pathway different from that by which PTH produces its effects. Transforming growth factor-beta (TGF-beta) enhanced responses to TNF; TGF-beta failed to inhibit the effects of TNF, even in long-term culture or when bones were pretreated with TGF-beta. Synergistic interactions between TNF and several other bone-resorbing factors have now been demonstrated. In contrast to the actions of TNF on certain other functions, the bone-resorbing effects of TNF, as determined in the fetal rat limb bone system, do not seem to be mediated by PAF, IL-1, or prostaglandins.

Animals↗
PubMed1992

Endothelin-1 actions on resorption, collagen and noncollagen protein synthesis, and phosphatidylinositol turnover in bone organ cultures.

The effects of endothelin-1 (ET) on several tissues are mediated by prostaglandins. In this study, we investigated the actions of ET on bone and determined whether they are mediated through prostaglandin-dependent pathways. Bone resorption, collagen, and non-collagen protein synthesis and inositol phosphate (IP) production were studied in neonatal mouse calvaria and fetal rat limb bone cultures. The effects of ET in the calvaria model were examined in the presence or absence of the cyclooxygenase inhibitor indomethacin (INDO). Bone resorption was stimulated by ET in the neonatal mouse calvaria, and this effect was inhibited by INDO. 45Ca release in the fetal rat limb bones was not affected by ET. ET stimulated collagen and noncollagen protein synthesis significantly in the calvaria model in the presence but not in the absence of INDO, suggesting that the anabolic effects of ET were masked by endogenous prostaglandin production. ET increased phosphatidylinositol turnover in both bone organ cultures. Although the addition of INDO reduced IP production slightly in the mouse calvaria, it was still significantly stimulated by ET. Our results demonstrate that ET has marked effects on bone tissue in vitro. Effects on resorption appear to be prostaglandin dependent, whereas the anabolic effects were not prostaglandin mediated. The stimulatory effects of ET on protein synthesis could be mediated through the IP signaling pathway. Since ET stimulates both bone resorption and anabolism, this peptide may have a role in the coupling of bone remodeling.

Animals↗
PubMed1991

Salmon stanniocalcin and bovine parathyroid hormone have dissimilar actions on mammalian bone.

Stanniocalcin (STC), a calcium-regulating glycoprotein hormone isolated from the corpuscles of Stannius of salmon, was tested for effects on bone and calcium metabolism in mammalian species (rats and mice). STC generally failed to alter serum calcium of parathyroidectomized rats at concentrations equimolar with effective concentrations of parathyroid hormone (PTH). STC did not increase cAMP in ROS 17/2.8 or UMR-108 osteosarcoma cells, OK kidney cells, fetal rat limb bones, or neonatal mouse calvariae, and similarly failed to increase urinary cAMP in rats. STC did not consistently stimulate resorption in any of the rodent bone culture systems, although variable resorptive responses were elicited in fetal mouse calvariae. The results indicate that this fish hormone has limited, if any, PTH-like activity on calcium metabolism in mammalian systems.

Animals↗
PubMed1990

Second messengers in thrombin-stimulated bone resorption.

Characterized human thrombins and two commercial bovine thrombin preparations were examined for their effects on bone resorption and on the cyclic AMP and phosphoinositide second messenger systems in bone. Human alpha- and gamma-thrombins, as well as both bovine thrombin preparations, stimulated bone resorption in vitro, whereas catalytically inactivated human diisopropylfluorophosphate (DIP)-alpha-thrombin did not significantly stimulate resorption. Human alpha-thrombin and a commercial bovine thrombin preparation increased cyclic AMP production in fetal rat limb bones, but another bovine commercial thrombin preparation and gamma-thrombin did not. Except for DIP-alpha-thrombin, all thrombins increased production of inositol phosphates in fetal rat limb bones at concentrations that stimulated resorption. In time course studies, bovine thrombin increased label in inositol trisphosphate at 30 s, with decreasing effects at later times. Inositol monophosphate increased progressively over 30 min. Our results are consistent with thrombin-stimulated bone resorption being mediated at least partially through the inositol phosphate pathway.

Animals↗