Emergence of viral hemorrhagic fevers: is recent outbreak of Crimean Congo Hemorrhagic Fever in India an indication?

The emerging and re-emerging diseases are posing a great health risk for the last few years. One such category of diseases is viral haemorrhagic fevers (VHFs), which have emerged in the new territories, worldwide. Crimean Congo Hemorrhagic Fever (CCHF) cases, for the first time in India, were reported from Gujarat, in January 2011. The emergence of diseases not reported earlier, pose great economic and social challenge, burden health system, and create panic reaction. Nonetheless, with recent experience in control of epidemic diseases, and advances in basic scientific knowledge; the public health community is better prepared for these unexpected events. This review provides information to physicians on CCHF for managing outbreak, and identifies public health measures to prevent emergence and re-emergence of VHFs (including CCHF) in future. The authors suggest that though, there are a few challenging and unanswered questions, the public health preparedness still remains the key to control emerging and re-emerging diseases. The countries where virus activities have been reported need to be prepared accordingly.

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor promotes endothelial cell survival through the activation of Akt/protein kinase B.

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor (KSHV-GPCR) is a key molecule in the pathogenesis of Kaposi's sarcoma, playing a central role in the promotion of vascular endothelial growth factor (VEGF)-driven angiogenesis and spindle cell proliferation. We previously have shown that KSHV-GPCR has oncogenic potential when overexpressed in fibroblasts and is responsible for the expression and secretion of VEGF through the regulation of different intracellular signaling pathways (A. Sodhi et al., Cancer Res., 60: 4873-4880, 2000; C. Bais et al., Nature, 391: 86-89, 1998). Here, we describe that this constitutively active G protein-coupled receptor is able to promote cell survival in primary human umbilical vein endothelial cells and that this effect is independent of its ability to secrete VEGF because it is not prevented by the expression of antisense constructs for VEGF or the addition of VEGF-blocking antibodies. Instead we found that ectopic expression of KSHV-GPCR potently induces the kinase activity of Akt/protein kinase B in a dose-dependent manner and triggers its translocation to the plasma membrane. This signaling pathway requires the function of phosphatidylinositol 3'-kinase and is dependent on betagamma subunits released from both pertussis toxin-sensitive and -insensitive G proteins. Furthermore, we found that KSHV-GPCR is able to protect human umbilical vein endothelial cells from the apoptosis induced by serum deprivation and that both wortmannin and the expression of a kinase-deficient Akt K179M mutant are able to block this effect. Finally, we observed that the Akt K179M protein also inhibits the activation of nuclear factor-KB induced by KSHV-GPCR, suggesting that this transcription factor may represent one of the putative downstream targets for Akt in the survival-signaling pathway. These results provide further knowledge in the elucidation of the signal transduction pathways activated by KSHV-GPCR and support its key role in promoting the survival of viral-infected cells. Moreover, the present findings also emphasize the importance of this G protein-coupled receptor in the development of KSHV-related neoplasias.

The Kaposi's sarcoma-associated herpes virus G protein-coupled receptor up-regulates vascular endothelial growth factor expression and secretion through mitogen-activated protein kinase and p38 pathways acting on hypoxia-inducible factor 1alpha.

The elucidation of the molecular mechanisms governing the transition from a nonangiogenic to an angiogenic phenotype is central for understanding and controlling malignancies. Viral oncogenes represent powerful tools for disclosing transforming mechanisms, and they may also afford the possibility of investigating the relationship between transforming pathways and angiogenesis. In this regard, we have recently observed that a constitutively active G protein-coupled receptor (GPCR) encoded by the Kaposi's sarcoma-associated herpes virus (KSHV)/human herpes virus 8 is oncogenic and stimulates angiogenesis by increasing the secretion of vascular endothelial growth factor (VEGF), which is a key angiogenic stimulator and a critical mitogen for the development of Kaposi's sarcoma. Here we show that the KSHV GPCR enhances the expression of VEGF by stimulating the activity of the transcription factor hypoxia-inducible factor (HIF)-1alpha, which activates transcription from a hypoxia response element within the 5'-flanking region of the VEGF promoter. Stimulation of HIF-1alpha by the KSHV GPCR involves the phosphorylation of its regulatory/inhibitory domain by the p38 and mitogen-activated protein kinase (MAPK) signaling pathways, thereby enhancing its transcriptional activity. Moreover, specific inhibitors of the p38 (SKF86002) and MAPK (PD98059) pathways are able to inhibit the activation of the transactivating activity of HIF-1alpha induced by the KSHV GPCR, as well as the VEGF expression and secretion in cells overexpressing this receptor. These findings suggest that the KSHV GPCR oncogene subverts convergent physiological pathways leading to angiogenesis and provide the first insight into a mechanism whereby growth factors and oncogenes acting upstream from MAPK, as well as inflammatory cytokines and cellular stresses that activate p38, can interact with the hypoxia-dependent machinery of angiogenesis. These results may also help to identify novel targets for the development of antiangiogenic therapies aimed at the treatment of Kaposi's sarcoma and other neoplastic diseases.

Expression and activation of lyn in macrophages treated in vitro with cisplatin: regulation by kinases, phosphatases and Ca2+/calmodulin.

Cisplatin [cis-dichlorodiammine platinum (II)], a potent chemoimmunotherapeutic drug, activates macrophages to tumoricidal state which is inhibited by protein tyrosine kinase(s) inhibitor. Cisplatin induces protein tyrosine phosphorylation of a number of cellular proteins suggesting the involvement of protein tyrosine kinase(s) in the activation process of macrophages. Therefore, the effect of cisplatin treatment on the expression and activation of lyn, a protein tyrosine kinase of src family, in macrophages was investigated. The underlying mechanism of lyn expression and activation was also analyzed. Cisplatin treatment increased lyn expression and activation in macrophages within 5 min of treatment. The expression and activation of lyn were observed to be biphasic processes in cisplatin-treated macrophages with the first peak appearing at 15 min and the second peak at 2 h of treatment. The appearance of second phase of lyn activation and second phase of lyn expression were two unrelated processes. The second peak of lyn activation was produced by the autocrine action of some soluble product(s) of cisplatin-treated macrophages, whereas the second phase of lyn expression was due to some intracellular factor. It was further observed that cisplatin-induced lyn expression and activation involves serine/threonine phosphatases 1/2A, protein tyrosine phosphatases, protein tyrosine kinase and protein kinase C. It was also observed that Ca2+/calmodulin and calmodulin-dependent kinases are involved in the regulation of cisplatin-induced lyn expression and activation in macrophages.

Expression of LFA-1 adhesion molecules on cisplatin-treated macrophages.

Appropriately activated mononuclear phagocytes mediate contact-dependent tumoricidal activity. Adhesion structures involved in contact-dependent tumor cytotoxicity have not been defined. The present study was aimed at identifying the adhesion structure involved in the tumoricidal activity of cisplatin-activated murine peritoneal macrophages. Tumor cells of different histological origin were used as targets in a 24-h cytotoxicity assay. Anti-CD18 (LFA-1 beta) substantially inhibited macrophage cytotoxicity whereas anti-LFA-1 alpha marginally inhibited macrophage-mediated cytotoxicity. When combined together, almost complete inhibition of tumoricidal activity was observed. Activated macrophages showed augmented binding to target cells and anti-LFA MAb inhibited the binding of resting and activated macrophages to target cells. Cisplatin augmented the expression of LFA-1 alpha and beta integrins and LPS had no effect as assessed by immunoprecipitation. These results implicate that in cisplatin activated macrophages LFA-1 alpha and beta integrins are important molecules in contact-dependent tumoricidal activity.

Synthesis, structure, and antitumor activity of N-salicyloyl-N'-(2-furylthiocarbonyl)hydrazine and its copper(II) complex.

N-Salicyloyl-N'-(2-furylthiocarbonyl)hydrazine (H2sfth) and its Cu(II) complex [Cu(sfth)] were prepared and characterized by physicochemical studies. The IR and ESR spectral studies imply dibasic tetradentate behavior of the ligand bonding through "thiolo" sulfur, enolic oxygen, and hydrazinic nitrogens in a polymeric structure. The electronic spectrum of the complex indicates a square-planar geometry around Cu(II). Maximum antitumor activity was observed when 25 mg/kg dose levels of H2sfth and Cu(sfth) were injected intraperitoneally in mice bearing either solid fibrosarcoma or ascites Dalton's lymphoma. However, H2sfth appeared to possess better antitumor activity as demonstrated by higher T/C (percent) values than those observed for Cu(sfth). The appearance of lymphocytes, leukocytes, and macrophages within the tumor mass 2-6 days after treatment are indicative of involvement of the host's immune system.

Production of interleukin-1 and tumor necrosis factor by bone marrow-derived macrophages: effect of cisplatin and lipopolysaccharide.

Mature macrophages derived in vitro from bone marrow progenitors under the influence of either L929 CM (a source of M-CSF) or GM-CSF have been shown to differ morphologically and functionally. Treatment of these bone marrow-derived macrophages with cisplatin or LPS resulted in the expression of enhanced tumoricidal activity and the production of significant amounts of extracellular and membrane-associated IL-1 and TNF. rGM-CSF-derived bone marrow macrophages produced higher amounts of TNF and IL-1 activity than L929CM-derived macrophages. Untreated bone marrow-derived macrophages showed little IL-1 and TNF activity. Bone marrow macrophages cultured with medium alone also did not respond to cisplatin or LPS for the production of IL-1 and TNF. Neutralization studies with anti-IL-1 and anti-TNF antibodies inhibited the IL-1 and TNF activity of bone marrow-derived macrophages. These results suggest that cisplatin or LPS treatment of murine bone marrow-derived macrophages results in increased expression of both released and membrane-associated IL-1 and TNF.

Increased production of interleukin-1 and tumor necrosis factor by human monocytes treated in vitro with cisplatin or other biological response modifiers.

Supernatants collected from cisplatin, lipopolysaccharide (LPS), muramyl dipeptide (MDP) or interferon-gamma (IFN-gamma) treated human monocytes enhance the thymocyte proliferation by a submitogenic concentration of concanavalin A. Also supernatants collected from cisplatin or IFN-gamma treated monocytes demonstrated enhanced cytotoxicity against actinomycin-D treated L 929 cells, suggesting that cisplatin or rIFN-gamma treated monocytes release tumor necrosis factor (TNF) into the culture medium. The supernatant collected from untreated monocytes showed only little IL-1 and TNF activity.

Up-regulation of IL-2 induced lymphokine activated killer cell activity by cisplatin and FK-565: involvement of calcium ion.

As reported earlier, the IL-2 induced lymphokine activated killer (LAK) activity is significantly up-regulated in the presence of cisplatin/FK-565. Based on these observations, we have investigated whether calcium is involved in the generation of LAK activity by IL-2 alone or along with CP/FK-565. We have shown that treatment of PBMC with IL-2 for four days caused an increase in intracellular free calcium and in ATP levels, which were further significantly enhanced when LAK cells were generated in the presence of CP/FK-565. Depletion of calcium resulted in decreased cytotoxic activity. Addition of tumor cells to LAK cells, generated in the presence of IL-2 alone or along with CP/FK-565 caused an instant rise in intracellular free calcium which was significantly decreased when an increase in intracellular free calcium was observed in calcium-free, EGTA-containing buffer. These data suggest that calcium is required for the activation and manifestation of lytic activity by LAK cells. Further, we observed that the increase in intracellular free calcium is not associated with the blastogenic response of peripheral blood mononuclear cells in response to treatment of IL-2 alone or together with CP/FK-565.

Effect of TNF priming of murine peritoneal macrophages on their activation to a tumoricidal state.

Murine peritoneal macrophages, on treatment with TNF (10 U/ml) for various durations, showed enhanced cyototoxicity against tumor target cells. The macrophage-mediated cytotoxicity was significantly enhanced when TNF-primed macrophages were treated with cisplatin, LPS, FK-565 or interferon-gamma for 24 h, compared to unprimed and treated macrophages. TNF-primed macrophages also showed enhanced expression of interleukin-1 and tumor necrosis factor activities on activation with different biological response modifiers.

Cisplatin-treated macrophages produce oncostatin M: regulation by serine/threonine and protein tyrosine kinases/phosphatases and Ca2+/calmodulin.

In the present study it was investigated whether cisplatin-treated murine peritoneal macrophages produce oncostatin M (OSM) and what is the underlying mechanism. The culture supernatants of cisplatin-treated macrophages significantly inhibited the proliferation of OSM-sensitive cell line A375. Within 15 min of cisplatin treatment significant OSM was synthesized and secreted by macrophages. Inhibitors of serine/threonine and protein tyrosine phosphatases augmented cisplatin-induced OSM production of macrophages. The protein kinase C and protein tyrosine kinase inhibitors significantly inhibited OSM production of cisplatin-treated macrophages. The OSM production of cisplatin-treated macrophages was also inhibited in the presence of Ca2+ chelators, Ca2+ channel blocker and calmodulin/calmodulin-dependent kinase inhibitors. These data suggest that OSM production of cisplatin-treated macrophages is regulated by opposing actions of phosphatases and kinases. It is also suggested that OSM production of cisplatin-treated macrophages is dependent on Ca2+, calmodulin and calmodulin-dependent kinase.

Mechanism of NF-kappa B translocation in macrophages treated in vitro with cisplatin.

In murine peritoneal macrophages cisplatin (cis-dichlorodiammine platinum (II)), a potent chemoimmunotherapeutic drug modulates the expression of several cytokines which contain nuclear factor kappa B (NF-kappaB) binding site suggesting the involvement of NF-kappaB in the activation process of macrophages by cisplatin. Therefore, we analyzed the effect of cisplatin treatment on NF-kappaB expression and activation in macrophages. The underlying mechanism of NF-kappaB translocation was also investigated. Cisplatin treatment increased cellular NF-kappaB content in macrophages treated for 60 min. NF-kappaB translocation was biphasic. Cisplatin-induced translocation of NF-kappaB took place within 5 min and reached its optimum by 15 min. A second phase of nuclear transfer of NF-kappaB was observed at 3 h of cisplatin treatment which was dependent on H2O2 production in cisplatin-treated macrophages. It was observed that cisplatin-induced NF-kappaB translocation involves serine/threonine phosphatases 1/2A, protein tyrosine phosphatases and genestein sensitive protein tyrosine kinase activities. H-7 sensitive protein kinase C do not fall in the signaling pathway of cisplatin leading to the translocation of NF-kappaB. Both cytosolic and membrane-associated factors were required for the induction of NF-kappaB translocation by cisplatin in macrophages.

Protein kinase C: a potential pathway of macrophage activation with cisplatin.

Cisplatin (CP) has been reported to activate murine macrophages to tumoricidal state, however, its mechanism of action is not known. In the present study it is reported that the production of: (a) interleukin-1 (IL-1); (b) tumor necrosis factor (TNF); (c) nitric oxide (NO); and (d) macrophage-mediated cytotoxicity by cisplatin-treated bone marrow-derived macrophages were inhibited by PKC inhibitors H-7 and chelerythrine chloride. Also, it was observed that treatment of macrophages with CP resulted in the translocation of PKC from the cytosol to the membrane fraction. These findings suggest the involvement of PKC in the activation of bone marrow-derived macrophages with cisplatin.

Activation of murine macrophages by tumor necrosis factor, interleukin-1, interferon-gamma and cisplatin.

Murine peritoneal macrophages were rendered tumoricidal to Dalton's lymphoma (DL) cells on incubation with recombinant tumor necrosis factor alpha (rTNF-alpha), recombinant interleukin-1 (rIL-1) and cisplatin in vitro. Simultaneous treatment of macrophages with suboptimal doses of rTNF-alpha and rIL-1 had additive effect on the activation of macrophages. Priming of macrophages with recombinant interferon gamma (rIFN-gamma) significantly enhanced the rTNF-alpha and rIL-1-induced macrophage cytotoxicity. Cisplatin was found to up-regulate rIL-1-induced macrophage activation but inhibited the activation of macrophages with rTNF-alpha. These studies indicate the potential of appropriate combination of these Biological Response Modifiers (BRMs) against neoplasia.

Up-regulation of induction of lymphokine (IL-2)-activated killer (LAK) cell activity by FK-565 and cisplatin.

The role of cisplatin and FK-565 in up-regulation of lymphokine-activated killer (LAK) cell induction by IL-2 was examined. Treatment of blood mononuclear cells (MNC) of healthy donors with cisplatin or FK-565 in the presence of IL-2 resulted in a significant increase in LAK activity against natural killer (NK)-resistant Daudi cells as assessed by the 4 h 51Cr release assay. Blood MNC treated with cisplatin alone was not cytotoxic to Daudi cells. However, MNC treated with FK-565 showed some cytotoxicity against Daudi cells. Addition of cisplatin to IL-2-stimulated MNC did not increase proliferation but did enhance cytotoxicity. FK-565 together with IL-2 increased both proliferation and cytotoxicity of blood MNC. These data suggest the potential of cisplatin and FK-565 in LAK adoptive immunotherapy for cancer treatment.

Impairment of T-cell functions with the progressive ascitic growth of a transplantable T-cell lymphoma of spontaneous origin.

It has been observed that the progressive ascitic growth of a transplantable T-cell lymphoma of spontaneous origin, designated Dalton's lymphoma (DL), in a murine host induces inhibition of various immune responses and is associated with an involution of thymus accompanied by a massive depletion of the cortical region and alteration in the distribution of thymocytes caused by tumour serum-dependent induction of apoptosis with a decrease of CD4(+)CD8(+), CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes. Here, we report that thymocytes of DL-bearing mice are defective in their proliferative ability and in their response to non-specific mitogenic stimulus in vitro. Also, antigen-specific T-cell proliferative ability representing the fundamental T(H) function declines under DL-bearing conditions and upon treatment with serum of DL-bearing mice. Moreover, a significant inhibition of T-cell cytolytic activity with a decreased ability to produce interferon gamma is shown by the T cells of DL-bearing mice and by the T cells treated with DL-ascitic fluid, DL-conditioned medium or serum of DL-bearing mice. Further, addition of interleukin-2 and anti-interleukin-10 to the cultures of thymocytes treated with serum of DL-bearing mice is found to inhibit the induction of apoptosis in thymocytes, a phenomenon associated with the progression of DL growth. Analysis of the results indicates an immune deviation with the predominance of a T(H2)-type response with the progression of tumour. We further discuss the possible mechanisms that may explain the observed tumour-induced diminution of T-cell immunity.

Activation of P388D1 macrophage cell line by chemotherapeutic drugs.

It has been found that certain antineoplastic drugs impart their function with a distinct duality. Besides being tumoricidal, they are capable of acting as immunopotentiator. This led us to investigate the effect of cytosine arabinoside (CA), vincristine sulphate (VS), cyclophosphamide (CS), mitomycin C (MMC), hydroxy urea (HU) and lipopolysaccharide (LPS) on a macrophage cell line P388D1. Supernatants collected from P388D1 cells treated with CA, VS, CS, MMC, HU or LPS demonstrated enhanced production of tumor necrosis factor (TNF) confirmed by bioassay on L929 tumor target cells and increased interleukin-1 (IL-1) production by standard thymocyte proliferation bioassay. Also, supernatants showed increased amounts of nitric oxide and lysozyme using Griess reaction and reduction in turbidity of Micrococcus lysodeikticus, respectively. The above findings demonstrate that these drugs may be used not only as chemotherapeutic agents but also as macrophage-activating agents.

Study on pinocytosis and antigen presentation by murine peritoneal macrophages to T cells in vitro after cisplatin treatment.

Macrophage monolayers showed significantly increased rate of pinocytosis on incubation with cisplatin (10 micrograms/ml). Further, the rate of pinocytosis was compared with macrophages treated with LPS (10 micrograms/ml). Cisplatin treated macrophages also showed enhanced capacity of antigen presentation to T cells in vitro. The antigen presenting capacity of cisplatin treated macrophages was compared with the antigen presenting capacity to LPS treated macrophages.

In vivo activation of murine peritoneal macrophages by intraperitoneal administration of cisplatin.

A single intraperitoneal injection of cisplatin (10 mg/kg body weight) in C3H/He mice increases the total number of peritoneal exudate cells (PEC) and macrophages (m phi) within 24 to 48 h. The total number of PEC from untreated mice ranged from 4 to 5 x 10(6) cells/ml containing 2.5 to 3 x 10(6) macrophages, whereas in cisplatin treated mice total number of PEC ranged up to 25 x 10(6) cells/ml. These PEC contained up to 16 x 10(6) m phi. The macrophages obtained from cisplatin injected mice show enhanced cytotoxicity, cytostasis and binding to Dalton's lymphoma cells in vitro. These activated macrophages release into the culture medium factors having cytolytic and cytostatic effect on Dalton's lymphoma cells. The activated macrophages also show enhanced capacity to release superoxide anions, hydrogen peroxide, lysozyme, arginase and interleukin-1.

Release of tumor cytolytic/cytostatic factor(s) by in vitro cisplatin treated macrophages: enhanced tumoricidal activity by increase in osmotic fragility of target cells.

In vitro cisplatin treatment of murine macrophages results in the release of tumor cytolytic/cytostatic factor(s) (TCF) into the culture medium. These cytolytic factors are cytotoxic/cytostatic only against Dalton's lymphoma and L929 tumor cells but not to normal splenocytes. The cisplatin-activated macrophages retain the capacity to lyse the tumor cells even after removal of the medium containing cisplatin. The mechanism of tumoricidal activity by the TCF was investigated. TCF containing medium was found to enhance the osmotic fragility of the tumor cells.