Sanjay Mammen Cherian

Novel mutations in the SDHD gene in pedigrees with familial carotid body paraganglioma and sensorineural hearing loss

Paraganglioma (PGL) is a rare disorder characterized by tumors of the head and neck region. Between 10% and 50% of cases of PGL are familial, and the disease is autosomal dominant and subject to age‐dependent penetrance and imprinting. The paraganglioma gene (PGL1) has been mapped to 11q22.3–q23, and recently germline mutations in the SDHD gene have been identified. The SDHD region contains another gene, DPP2/TIMM8B, the homolog of which causes dystonia and deafness seen in Mohr‐Tranebjaerg syndrome. Using four PGL pedigrees, two of which exhibit coinheritance of PGL and sensorineural hearing loss or tinnitus, analysis of 14 microsatellite markers provided support for linkage to the PGL1 locus. Sequence analysis identified novel mutations in exon 1 and exon 3 of the SDHD gene, including a novel two base pair deletion in exon 3 creating a premature stop codon at position 67; a novel three base pair deletion in exon 3 resulting in the loss of Tyr‐93; a missense mutation in exon 3 resulting in the substitution of Leu‐81 for Pro‐81; and a novel G‐to‐C substitution in exon 1 resulting in the substitution of Met‐1 for Ile‐1. No base changes were detected in the DPP2/TIMM8B gene. There was no apparent loss of heterozygosity at the site of the SDHD mutations. However, RT‐PCR analysis of tumor samples showed monoallelic expression of the mutant (paternal) allele as expected for imprinting. This has not previously been shown for this disorder. The inheritance and expression of the SDHD gene is consistent with the PGL1 gene being subject to genomic imprinting.

Induction of apoptosis in vascular cells by plasminogen activator inhibitor-1 and high molecular weight kininogen correlates with their anti-adhesive properties.

Abstract Plasminogen activator inhibitor-1 (PAI-1) and twochain high molecular weight kininogen (HKa) exert antiadhesive properties in vitronectindependent cell adhesion. Here, the hypothesis was tested that these anti-adhesive components promote apoptosis in vascular cells. PAI-1 or HKa induced a 2- to 3-fold increase in apoptosis of human umbilicalvein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC) adherent to vitronectin, as determined by annexin VFACS assay, similar to αvintegrin inhibitor cyclo-(Arg-Gly-Asp-D-Phe-Val)-peptide (cRGDfV). Apoptosis occurred after 12 h incubation and was attributable to caspase 3 activation that in turn induced DNA fragmentation. Induction of apoptosis strongly correlated with the antiadhesive effect of PAI-1 and HKa on these cells. In contrast, PAI-1 and HKa did not affect fibronectin-dependent adhesion or cell survival. uPA did not influence apoptosis in vitronectin- or fibronectin-adherent cells. In atherosclerotic vessel sections, congruent distribution of vitronectin, PAI-1, HK, and of components of the urokinase plasminogen activator/receptor system with apoptotic cells lining foam cell lesions was demonstrated by immunostaining. These results indicate that inhibition of vitronectindependent cell adhesion through PAI-1 and HKa correlates with apoptosis induction in vascular cells mediated through the caspase 3 pathway. Co-distribution of apoptosis with plasminogen activation system components in atherosclerosis exemplifies the significance of antiadhesive mechanisms and apoptosis for tissue remodeling, such as in neointima development.

Increased expression of disintegrin-metalloproteinases ADAM-15 and ADAM-9 following upregulation of integrins α5β1 and αvβ3 in atherosclerosis

Regulation of αvβ3 and α5β1 integrin function plays a crucial role in atherosclerosis. Possible regulators of integrin–matrix interactions are integrin‐binding ADAMs (proteins with a disintegrin‐ and metalloproteinase‐domain), like ADAM‐15 and ADAM‐9. Molecular interactions between ADAM‐15, α5β1, and αvβ3 have been demonstrated. ADAM‐9 and ADAM‐15 were found to be interdependently regulated. This study, therefore, investigated whether the upregulation of integrins α5β1 and αvβ3 was correlated with the expression of integrin‐binding ADAMs in atherosclerotic processes. Human arterial and venous vascular smooth muscle cells (VSMCs) were incubated with PDGF over different time intervals up to a 3‐day culture period. mRNA concentrations, quantified by real‐time RT‐PCR and normalized to PBGD, of integrins αvβ3 and α5β1 were strongly increased after a 12‐h PDGF‐incubation in arterial and venous VSMC. ADAM‐15 and ADAM‐9 mRNA production showed a corresponding increase following integrin upregulation after a 24‐h incubation period. Western blot anaylsis revealed an increased protein expression of integrins and ADAMs in PDGF‐stimulated VSMC. Additionally, mRNA concentrations of atherosclerotic and normal human specimens were quantified by real‐time RT‐PCR. mRNA of ADAMs and integrins was significantly increased in atherosclerotic arteries compared to normal arteries. Immunohistochemistry of these specimens showed an increased expression and codistribution of both ADAMs and integrins in atherosclerosis. In conclusion, upregulation of ADAM‐15 and ADAM‐9 in atherosclerosis appears to follow an increase in α5β1 and αvβ3 integrins. Since α5β1 and αvβ3 are known to promote smooth muscle cell migration and proliferation, upregulation of ADAM‐15 and ADAM‐9 could balance integrin–matrix interactions and cell migration, thus modulating neointima progression. J. Cell. Biochem. 89: 808–823, 2003.

Accumulation of lymphocytes, dendritic cells, and granulocytes in the aortic wall affected by Takayasu's disease.

The aim of this study was to analyze the cellular composition of the arterial wall in Takayasus disease and to investigate the contribution of the various cell types to the immunoinflammatory processes and degenerative alterations of the vessel wall in this disease. Specimens of aorta were obtained at operation from 10 patients with Takayasus arteritis. The duration of disease ranged from 2 months to 13 years. Immunohisto chemical investigation was carried out using the antibodies CD3 (to identify T-cells), CD20 (B-cells), S-100 (dendritic cells), CD68 (macrophages), CD15 (granulocytes), von Willebrand factor (endothelial cells), and alpha-smooth muscle actin (smooth muscle cells). All specimens showed distinctive histologic features of Takayasus arteritis and contained inflammatory infiltrates, but the degree of their accumulation within the aortic wall varied. Inflammatory infiltrates within the deep part of the intima, around areas of neovascularization and within the adventitia contained T-cells colocalizing with dendritic cells. Nodules formed by large numbers of intermingling T-cells and B-cells enriched with dendritic cells were observed in the adventitia. Massive accumulation of granulo- (continued on next page) cytes and their destruction within the adventitia were prominent in all cases. This is the first study that establishes the presence of dendritic cells and granulocytes in Takayasus disease. Dendritic cells are probably involved in the immunoinflammatory processes through their interaction with T-cells and B-cells. The present observations may help understanding of the pathogenesis of Takayasus disease.

Immunohistochemical and ultrastructural evidence that dendritic cells infiltrate stenotic aortocoronary saphenous vein bypass grafts

We earlier speculated that antigen-presenting dendritic cells may be involved in the immune reactions leading to saphenous vein bypass graft failure. The purpose of this study was to confirm whether dendritic cells are present in stenotic human saphenous vein bypass grafts. Segments of stenotic saphenous vein grafts were explanted from 14 patients at re-do bypass operation and ten normal saphenous veins were harvested during femoro-popliteal grafting. Sections of specimens were analysed using cell type specific antibodies to identify dendritic cells (CD1a, S-100), T-lymphocytes (CD3), macrophages (CD68), smooth muscle cells (alpha-SMA) and endothelial cells (FVIII). Dual immunostaining, confocal immunofluorescent laser scanning microscopy and electron microscopy were used. Stenotic grafts showed structural alterations of intimal hyperplasia and varying degrees of atherosclerotic degeneration. No cells expressing CD1a and S-100 were observed in the intima and media of normal saphenous veins. Cells expressing these antigens were present around areas of medial neovascularization and within intimal atherosclerotic lesions in saphenous vein bypass grafts. Electron microscopy demonstrated the presence of cells containing a well-developed tubulovesicular system which is unique to cells from the dendritic cell family. Double immunohistochemistry and confocal immunofluorescent microscopy revealed the co-localization of T-lymphocytes with dendritic cells. Dendritic cells are present in stenotic saphenous vein bypass grafts. Dendritic cells may be responsible for antigen presentation and modulation of immune reactions in accelerated graft atherosclerosis through their interaction with T-lymphocytes.

Dendritic cells in venous pathologies.

Dendritic cells are potent antigen-presenting cells responsible for the activation of T-lymphocytes in various immune responses. Their role in the initiation of immune reactions in allergies, autoimmune diseases, tumors, transplantation, and, more recently, in atherosclerosis has been well established, but their involvement in venous pathologies has not been previously investigated. The aim of this study was to determine whether dendritic cells are present in veins affected by varicosity and thrombophlebitis. Three groups of veins obtained at operation were studied: (1) varicose veins of the great saphenous vein from patients who were undergoing vein stripping for primary varicosity; (2) segments of the great saphenous vein from patients with varicosity complicated by thrombophlebitis; and (3) great saphenous veins without varicosity or thrombophlebitis from patients who were undergoing femoropopliteal bypass grafting. The specimens were fixed in 10% neutral buffered formalin and embedded in paraffin, and the sections were stained with antibodies to S-100 (to identify dendritic cells), CD3 (T-lymphocytes), CD68 (macrophages), von Willebrand factor (endothelial cells), alpha-smooth muscle actin (smooth muscle cells), and CD15 (mast cells) by use of avidin-biotin complex (ABC) immunoperoxidase technique. Immunohistochemical examination showed that no S-100 positive dendritic cells were present in normal saphenous veins. In contrast, S-100- positive cells with dendritic cell morphology were detected in the intima and media of veins with varicosity and thrombophlebitis, where they represented a minor cell popula tion. S-100-positive dendritic cells were located between smooth muscle cells as well as around areas of neovascularization where they colocalized with T-lymphocytes. The present work suggests that dendritic cells might be involved in pathological processes in veins affected by varicosity and thrombophlebitis. The authors speculate that dendritic cells may be involved in the inflammatory mechanisms in these veins through their inter action with T-lymphocytes.

Colonisation of prosthetic grafts by immunocompetent cells in a sheep model

The present study examined the distribution of immunocompetent cells in synthetic vascular grafts in an experimental sheep model. Sixty-two adult Merino sheep underwent synthetic patch closure of a longitudinal arteriotomy in the left common carotid artery. The synthetic patch materials used were gelatin sealed Dacron (n=10), fluoropassivated Dacron (n=10), Fluoropassiv (n=12), polyurethane (n=10), expanded polytetrafluoroethylene (n=10) and carbon-lined expanded polytetrafluoroethylene (n=10). The sheep were sacrificed after four weeks when the prosthetic patches were harvested and fixed in 10% neutral buffered formalin. Transverse sections were taken along the graft and paraffin embedded. Serial sections were stained with cell type specific antibodies to identify T-lymphocytes (CD3(+)), dendritic cells (S-100(+)), endothelial cells (von Willebrand factor(+)) and smooth muscle cells (smooth muscle alpha-actin(+)). All six graft types contained CD3(+) and S-100(+) cells in the neointima, within the synthetic matrix and in the perigraft layer. Three different tissue responses to synthetic materials were observed and the grafts were classified accordingly into three groups: (1) gelatin sealed Dacron, fluoropassivated Dacron and Fluoropassiv; (2) expanded polytetrafluoroethylene and carbon-lined expanded polytetrafluoroethylene; (3) polyurethane. The three synthetic materials in Group 1 showed almost identical reactions with least accumulation of immunocompetent cells within the synthetic material but greater accumulation of immuno-inflammatory infiltrates in the perigraft vascular tissue. In this group, new vessels penetrated into the synthetic material and there was prominent formation of foreign body (giant) cells. Group 2 showed greater accumulation of immunocompetent cells within the synthetic material itself but only sparse immuno-inflammatory infiltrates in the perigraft tissue. Group 3 showed a high degree of inflammatory response within both the synthetic material and the perigraft vascular tissue. These observations demonstrate that immunocompetent cells colonise the synthetic matrix of grafts and accumulate in the perigraft tissue, but inflammatory responses vary in different graft types.

Development of an antidiabetic formulation (ADJ6) and its inhibitory activity against α-amylase and α-glucosidase

There has recently been much advancement in the diagnosis, treatment, and research of metabolic disorders, especially diabetes. Current research around the world is focused on finding an alternative source of treatment from natural resources for diabetic management, apart from the available synthetic medicines. The present study is a preliminary study of a polyherbal formulation using edible natural resources and an assessment of its antidiabetic activity. The formulation was screened for its phytochemical constituents, total phenols, flavonoids, and vitamin C content. It was also analyzed for its inhibitory effect against the digestive enzymes α-amylase and α-glucosidase, compared with the standard drug acarbose. The formulation showed the presence of major constituents such as steroids, cardiac glycosides, phenols, flavonoids, and saponins. It also had a high level of phenols (340 ± 2.5 mg/g), flavonoids (235.4 ± 8.3 mg/g), and vitamin C (470.8 ± 16.6 mg/g), and showed a half-maximal inhibitory concentration (IC50) value of 0.41 ± 0.03 mg/mL and 0.51 ± 0.01 mg/mL for amylase and glucosidase, respectively. The results showed that ADJ6 had a significant inhibitory activity on α-amylase and α-glucosidase; however, its inhibitory activity was less than that of acarbose. The plants that are formulated in ADJ6 possess potent antidiabetic activity. Thus, we found that ADJ6 is a potent lead for effective diabetic management; however, an evaluation of the formulation must be illustrated using an in vivo model.

Development of an antidiabetic polyherbal formulation (ADPHF6) and assessment of its antioxidant activity against ROS-induced damage in pUC19 and human lymphocytes – an in vitro study

Abstract Background: Polyherbalism, an alternative natural-based therapy for various disorders, has been quoted about 1,300 years before in Sharangdhar Samhita. Herbal-based combination therapy stages a vital role for the treatment of type 2 diabetes mellitus (T2DM) and associated complications. The present study aims at developing an Ayurvedic-based polyherbal formulation (ADPHF6) and the assessing its antidiabetic and antioxidant property. Methods: ADPHF6 polyherbal formulation was measured for phytochemical components by qualitative methods. The polyherbal formulation was quantitatively estimated for its phytochemical constituents, i. e. total phenol and flavonoid content. Further, the antioxidant property of ADPHF6 formulation was evaluated by 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging assay, hydrogen peroxide radical scavenging assay and metal chelating assay. α-Amylase and α-glucosidase inhibitory activities of polyherbal formulation were also assessed. ADPHF6 was further analysed for its protective antioxidant property against reactive oxygen species (ROS‾)-induced damage in human lymphocyte DNA and pUC19 plasmid. Results: ADPHF6 polyherbal formulation revealed the presence of phytochemical constituents such as alkaloids, flavonoids, phenols, tannins, terpenoids, saponins and cardiac glycosides in significant levels. Further, it also measured the higher levels of total phenols (473.3±3.05 mg/g) and flavonoid (664±5.29 mg/g) content. Polyherbal formulation also exhibited IC50 values of 49.9±0.15, 65.1±0.10 and 60.1±0.05 mg/mL for 2,2- diphenyl-1-picryl-hydrazyl-hydrate (DPPH), hydrogen peroxide (H2O2) and Fe2+ radical scavenging activities, respectively. ADPHF6 revealed an inhibitory activity (IC50) of 0.67±0.01 and 0.81±0.01 mg/mL for α-amylase and glucosidase, respectively. Pre-treated human peripheral blood lymphocytes with ADPHF6 aqueous extract illustrated enhanced protection against ROS-mediated damage as compared with post-treated groups. DNA nicking assay rendered protective activity against the OH¯ radical-induced DNA damage in supercoiled pUC19 plasmid. Conclusions: Our present study demonstrates that ADPHF6 offers potent inhibitory activity against free radicals as well as digestive enzymes. However, studies should be conducted using in vivo model to further elucidate the effect against free radicals and its anti-hyperglycaemic activity in the management of non-insulin-dependent diabetes.

A rare case of life-threatening spontaneous psoas hematoma following cardiac surgery

Spontaneous psoas haematomas are uncommon, even in patients with coagulopathies on anticoagulation therapy. We report a unique case of a life-threatening spontaneous psoas haematoma in a patient during the immediate post-operative period following open heart surgery, despite a normal pre- and post-operative coagulation profile.