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.

Synthesis, characterization and antiproliferative activity of 1,2-naphthoquinone and its derivatives.

In the present study substituted 1,2-naphthoquinones were synthesized, purified and characterized by spectroscopic studies (UV, FT-IR, ¹H NMR, ¹³ C NMR and elemental analysis). These compounds were evaluated for cytotoxicity against a panel of human cancer cell lines (Hep-G2 for liver sarcoma, MG-63 for osteosarcoma and MCF-7 for human breast cancer). The cells were dosed with these ortho-naphthoquinone derivatives at varying concentrations, and cell viability was measured by a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay with doxorubicin as positive control. Significant anticancer activities were observed in vitro for some members of the series, and compounds 1,2-naphthoquinone 2-thiosemicarbazone, 1,2-naphthoquinone-2-semicarbazone, 4-amino-1,2-naphthoquinone 2-thiosemicarbazone and 4-amino-1,2-naphthoquinone-2-semicarbazone are active cytotoxic agents against different cancer cell lines with IC50 values in the range of 5.73-17.67 µM. The obtained data suggested that better anticancer activity was linked with introduction of thiosemicarbazone and semicarbazone moiety in 1,2-naphthoquinone ring system. Outcomes of experimentation also reveal that incorporation of amino group in 1,2-naphthoquinone moiety contributes positively for cytotoxic action of compounds. Docking experiments showed a good correlation between their calculated interaction energies with the topoisomerase-II and the observed IC50 values of all these compounds.

Midlife predictors of Alzheimer's disease.

Factors contributing to increased risk for Alzheimer's disease (AD) include age, sex, genes, and family history of AD. Several risk factors for AD are endogenous; however, accumulating evidence implicates modifiable risk factors in the pathogenesis of AD. Although the continued task of identifying new genes will be critical to learning more about the disease, several research findings suggest that potentially alterable environmental factors influence genetic contributions, providing targets for disease prevention and treatment. Here, we review midlife risk factors for AD, and address the potential for therapeutic intervention in midlife.

Effect of monocyte chemoattractant protein-1 on murine bone marrow cells: proliferation, colony-forming ability and signal transduction pathway involved.

Monocyte chemoattractant protein-1 (MCP-1) plays a crucial role in the migration and activation of leukocytes in both physiological and pathological contexts. In this paper, we report the in vitro effect of MCP-1 on myeloid haematopoiesis. MCP-1-treated murine nonadherent bone marrow cells (NABMCs) were assayed for in vitro proliferation and colony forming ability. It is observed that MCP-1 treatment in vitro caused an enhancement in the proliferation and colony forming ability of the murine NABMCs as compared to the untreated cells. This response was concentration-dependent and most effective at a dose of 100ng/ml MCP-1. In the presence of MCSF (200U/ml), GCSF (200U/ml), GMCSF (200U/ml) or IL-3 (200U/ml), the MCP-1-induced colony forming ability of the NABMCs was significantly augmented, indicating a synergistic effect of MCP-1 with these CSFs. However, irrespective of the CSFs used, MCP-1 stimulated the lineage-restricted differentiation of the murine BMCs into predominantly the granulocytic lineage. NABMCs cultured in medium alone formed minimal colonies. The probable signal transduction mechanism responsible for the MCP-1-induced NABMC proliferation/differentiation was also investigated. The results of the colony forming assay indicate that the protein kinase inhibitors, genistein (10&mgr;g/ml), chelenthryin chloride (10&mgr;M), wortmannin (200nM) and PD98059 (10&mgr;M) significantly blocked the in vitro colony forming ability of the MCP-1-treated NABMCs, while the phosphatase inhibitors, okadaic acid (10nM) and sodium orthovanadate (10&mgr;M) caused an increase in the BMC colony forming ability in response to MCP-1. These data suggests the involvement of the respective protein kinases and phosphatases in the above process. Correlating with this, the role of several signaling molecules likes Lyn, p42/44MAPK, PI3K and STAT5 has also been implicated in the signal cascade of murine NABMC proliferation/differentiation following MCP-1 treatment.

Regulation of nitric oxide production by murine peritoneal macrophages treated in vitro with chemokine monocyte chemoattractant protein 1.

Monocyte chemoattractant protein 1 (MCP-1) is an important mediator of monocyte/macrophage recruitment and activation at the sites of chronic inflammation and neoplasia. In the current study, the role of nitrogen monoxide (NO) in the activation of murine peritoneal macrophages to the tumoricidal state in response to in vitro MCP-1 treatment and the regulatory mechanisms involved therein were investigated. Murine peritoneal macrophages upon activation with MCP-1 showed a dose- and time-dependent production of NO together with increased tumoricidal activity against P815 mastocytoma cells. N-monomethyl-l-arginine (L-NMMA), a specific inhibitor of the l-arginine pathway, inhibited the MCP-1-induced NO secretion and generation of macrophage-mediated tumoricidal activity against P815 (NO-sensitive, TNF-resistant) cells but not the L929 (TNF-sensitive, NO-resistant) cells. These results indicated l-arginine-dependent production of NO to be one of the effector mechanisms contributing to the tumoricidal activity of MCP-1-treated macrophages. Supporting this fact, expression of iNOS mRNA was also detected in the murine peritoneal macrophages upon treatment with MCP-1. Investigating the signal transduction pathway responsible for the NO production by the MCP-1-activated murine peritoneal macrophages, it was observed that the pharmacological inhibitors wortmannin, H-7 (1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride), and PD98059 blocked the MCP-1-induced NO production, suggesting the probable involvement of phosphoinositol-3-kinase, protein kinase C, and p42/44 MAPkinases in the above process. Various modulators of calcium and calmodulin (CaM) such as EGTA, nifedipine, TMB-8 (3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester), A23187, and W-7 (N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide) were also found to modulate the in vitro macrophage NO release in response to MCP-1. This observation indicated the regulatory role of calcium/CaM in the process of MCP-1-induced macrophage NO production. Similarly, the role of serine/threonine and protein tyrosine phosphatases in the above pathway was suggested using the specific inhibitors of these phosphatases, okadaic acid and sodium orthovanadate.

MAPK and Akt act cooperatively but independently on hypoxia inducible factor-1alpha in rasV12 upregulation of VEGF.

Oncogenic ras upregulates the expression of VEGF through the activation of the transcriptional enhancer hypoxia inducible factor-1alpha (HIF-1alpha) by a still poorly understood mechanism. Here, we demonstrate that both the Raf/MEK/MAPK and the PI3 kinase/Akt signaling pathways potently and additively stimulate the expression from a hypoxia response element (HRE) within the 5'flanking region of the VEGF promoter. Interestingly, while MAPK appears to specifically upregulate the transactivation activity of HIF-1alpha through direct phosphorylation of its regulatory/inhibitory domain, GSK-3, a downstream target of Akt, directly phosphorylates the HIF-1alpha oxygen-dependent degradation domain. These results suggest a novel mechanism whereby two divergent signaling pathways emerging from Ras may cooperatively but independently regulate the activity of a HIF-1alpha, thereby promoting the expression of a potent angiogenic mediator.

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.

Ascitic growth of a spontaneous transplantable T cell lymphoma induces thymic involution. 2. Induction of apoptosis in thymocytes.

It has been observed that the progressive ascitic growth of a transplantable T cell lymphoma of spontaneous origin, designated as Dalton's lymphoma (DL), induces inhibition of various immune responses and is associated with an involution of the thymus accompanied by a massive depletion of the cortical region and alteration in the distribution of thymocytes, with a decrease in CD4+CD8+, CD4+CD8- and CD4-CD8+ thymocytes. Morphological evaluation of thymocytes from DL-bearing mice revealed that with the progression of DL, a majority of thymocytes exhibited morphological features characteristic of apoptotic cell death, which included contracted cell bodies, condensed, uniformly circumscribed and densely stained chromatin, and membrane-bound apoptotic bodies containing one or more nuclear fragments. Quantitative and qualitative analysis of the DNA extracted from the thymocytes of DL-bearing mice revealed DNA fragmentation that increased concomitantly with the progression of DL and showed an oligonucleosomal DNA ladder pattern upon agarose gel electrophoresis, a hallmark of apoptotic cell death. Attempts to identify apoptotic factor(s) showed that the serum of DL-bearing mice contained certain soluble factor(s) that augmented the induction of apoptotis in thymocytes in a time- and dose-dependent manner. Although DL cells or their products, such as DL-cell-conditioned medium or DL-cell-free ascitic fluid, could also induce apoptosis of thymocytes in vitro, the magnitude of the same was consistently lower than that induced by the serum of DL-bearing mice. Further, elucidation of the mechanism of apoptosis induction in thymocytes with respect to the involvement of apoptosis-related genes revealed that the death pathway followed an interleukin-1 beta-converting-enzyme-dependent, Fas-mediated apoptotic cascade, with a concomitant increase in the protein products of the bax, bad, p53, fas and fasL genes and cleavage of the 23-kD N-terminal fragment of Bcl-2 that exhibited Bax-like death effector properties.

Ascitic growth of a spontaneous transplantable T cell lymphoma induces thymic involution. 1. Alterations in the CD4/CD8 distribution in thymocytes.

We have previously shown that the progressive ascitic growth of a transplantable T cell lymphoma of spontaneous origin in a murine host, designated as Dalton's lymphoma (DL), induces the inhibition of various immune responses. In a quest to understand the mechanism(s) of tumor-growth-dependent immunosuppression, we were interested to investigate if the thymus, the center for the differentiation of immunocompetent T cells, undergoes any alteration concomitant with the growth of DL. Thus, DL was grown as an ascitic tumor in BALB/c mice for a period of 4 or 17 days, designated as the early and late tumor stages, respectively, and the thymuses were examined immediately after sacrifice of the animals on the 4th or 17th day of tumor transplantation. Progressive growth of DL was observed to be associated with thymic atrophy, as well as an involution of thymic organization and a depletion of cell mass. Histological sections of thymus from DL-bearing mice revealed a complete disintegration of the thymic architecture with a massive depletion of the cortical region and disappearance of the corticomedullary junctions. Flow cytometric analysis of alterations in the distribution of thymocytes revealed a decrease in CD4+CD8-, CD4-CD8+ and CD4+CD8+ cell populations, whereas the CD4-CD8- population showed an increase, suggesting an impairment in thymocyte differentiation at an early T cell maturation stage. Furthermore, tumor growth was shown to suppress the proliferation ability of thymocytes. Moreover, an increase in thymocytes of smaller size was also found with the progression of DL, which is an indication that a large fraction of thymocytes of a small, abnormal size could be apoptotic cells. Furthermore, the paper discusses the immunological implications of thymic atrophy in a host bearing a T cell lymphoma.

Anticancer drug-induced apoptosis in human monocytic leukemic cell line U937 requires activation of endonuclease(s).

Anticancer agents effect tumor cell killing both in vivo and in vitro through the induction of apoptosis. Endonuclease-mediated internucleosomal DNA fragmentation, the most widely used biochemical marker of apoptosis, has been shown to play a central role in apoptosis in many experimental systems. In the present investigation, we report that activation of endonuclease(s) leading to oligonucleosomal DNA fragmentation is common and an essential event in apoptosis, induced by different anticancer drugs, adriamycin, etoposide and cisplatin. The endonuclease inhibitors aurintricarboxylic acid and zinc ion prevented apoptotic cell death in human monocytic leukemic cell line U937, as documented by DNA fragmentation, morphological and nuclear alterations, and cell viability assay. Additional studies suggest endonuclease(s)-mediated DNA fragmentation may not play a central role in apoptosis in the same cell line in response to other inducers such as heat shock and cells may undergo cell death showing all morphological features of apoptosis even in the absence of DNA fragmentation.

Mechanism of thymocyte apoptosis induced by serum of tumor-bearing host: the molecular events involved and their inhibition by thymosin alpha-1.

The observations presented in this paper indicate that serum of Dalton's lymphoma (DL) bearing mice contained certain soluble factor(s) that augmented the induction of apoptosis in thymocytes in a time- and dose-dependent manner. DL-ascitic fluid and DL-conditioned medium could also induce apoptosis of thymocytes in vitro, though the magnitude of the same was consistently lower than that induced by serum of DL-bearing mice. It was observed that the interaction of FasL and TNFalpha with their respective receptors could trigger apoptosis in thymocytes. Elucidation of the signal transduction mechanism revealed involvement of protein tyrosine kinase, protein kinase C and ser/thr phosphatases with concomitant increase in the level of protein products of apoptosis associated genes p53, bax, bad, fas and fas ligand and cleavage of N-terminal 23 kDa fragment of Bcl-2 that exhibited Bax-like death effector properties. Further, we report, for the first time, the ability of thymosin alpha-1, an immunopotentiating thymic hormone, to antagonize apoptosis in thymocytes induced by factors present in serum of DL-bearing mice. The underlying mechanism of tumor serum induced apoptosis inhibition by thymosin alpha-1 was also analyzed. The signal transduction cascade evoked by thymosin alpha-1 involves activation of protein kinase C with a decrease in the level of protein products of proapoptotic genes like bax and bad and increase in the protein products of bcl-2 gene.

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 murine bone-marrow derived macrophages in vitro with Thymosin-alpha-1 to tumoricidal state: a comparative study on normal and tumour-bearing hosts.

The present investigation establishes the ability of Thymosin alpha l (T alpha l) to activate murine bone-marrow derived macrophages (BMDMs) in vitro to tumoricidal state with concomitant release of NO, TNFalpha and IL-1. The T alpha l-induced cytotoxicity and the secretion of soluble lytic factors were both dose- and time dependent. BMDMs cultured from the Dalton's Lymphoma bearing mice (DL-BMDMs) exhibited reduced cytolytic activity towards DL-tumour target cells on activation with T alpha l as compared to the BMDMs obtained from normal mice (N-BMDMs). The DL-BMDMs displayed enhanced TNFalpha and IL-1 release as compared to the N-BMDMs when treated with T alpha l. On the other hand, it is observed that the production of NO and the expression of iNOS was higher in the N-BMDMs as compared to the DL-BMDMs on treatment with T alpha l. Although T alpha l could trigger the tumoricidal functions of BMDMs from normal and DL-tumor bearing hosts, the progressive growth of DL-tumour in ascitic form leads to an alteration in the antitumour response of macrophages. These observations further suggest that a disregulation in the production of inflammatory cytokines like TNF-alpha, IL-1 and the inhibition of NO production in response to DL growth may mutually contribute in explaining the tumour-induced immunosuppression as observed in the DL-bearing mice.

Prior immunity to a carrier enhances antibody responses to hCG in recipients of an hCG-carrier conjugate vaccine.

Pre-sensitization with carrier often leads to epitopic suppression of subsequent anti-hapten antibody responses. To ascertain whether epitopic suppression occurs in humans, we examined the effect of pre-existing anti-carrier immunity on antibody responses to hCG in volunteers of a clinical trial of an hCG-based conjugate birth-control vaccine. When we studied the correlation between pre-vaccination anti-carrier immunity and post-vaccination anti-hCG responses, we found that prior immunity to the carriers did not lead to epitopic suppression of anti-hCG responses. On the contrary, it was found that prior immunity to TT, one of the two carriers used in this vaccine, led to enhancement of anti-hCG responses. Our data indicates that prior immunity to the carriers may not be detrimental to the performance of conjugate vaccines, and may actually be beneficial in some cases.

Expression and activation of RAS and mitogen-activated protein kinases in macrophages treated in vitro with cisplatin: regulation by kinases, phosphatases and Ca2+/calmodulin.

Cisplatin (cis-dichlorodiammineplatinum II), a potent antitumour compound, stimulates immune responses by activating monocytes/macrophages and other cells of the immune system. However, the exact mechanism by which cisplatin activates these cells is poorly characterized and attempts are being made to understand this mechanism. Previous studies from this laboratory have shown that Lyn, a protein tyrosine kinase of the src family, and nuclear factor (NF)-kappaB are involved in cisplatin-induced macrophage activation. Recent studies suggest that the RAS and mitogen-activated protein (MAP) kinases function as a connecting link between activated lyn and NF-kB, which raises the possibility of their involvement in cisplatin-induced macrophage activation. Therefore, this study was undertaken to investigate the effect of cisplatin treatment on the expression/activation of RAS (a low molecular weight GTP-binding protein) and MAP kinases in murine peritoneal macrophages. The underlying mechanism of expression/activation of RAS and MAP kinases in cisplatin-treated macrophages was also investigated. Immunoblotting and immune-complex kinase assays revealed that cisplatin treatment of macrophages leads to increased expression/activation of RAS and MAP kinases, with optimal expression/activation at 15 min of treatment. Using a battery of specific inhibitor/modulators of different signalling molecules, this study shows that expression and activation of MAP kinases are two unrelated processes. It was also observed that kinase (protein tyrosine and protein kinase C) inhibitor and Ca2+/calmodulin antagonist inhibit expression/activation of RAS/MAP kinases in macrophages, whereas phosphatases (protein tyrosine and serine/threonine) inhibitor up-regulate these kinases.

Age-dependent alterations in the tumoricidal functions of tumor-associated macrophages.

Age-dependent tumor cytolytic functions of tumor-associated macrophages (TAM) obtained from mice bearing different stages of Dalton's lymphoma (DL), a spontaneous T cell lymphoma, were studied. Mice were separated into three groups on the basis of their reproductive status as indicator of age: young (prereproductive); adult (reproductive) and old (postreproductive). DL was injected (1 x 10(5) cells/mouse) intraperitoneally in mice; days 4, 10 and 16 from the day of injection were referred to as early, mid and late tumor stages, respectively. Normal peritoneal macrophages and macrophages isolated from the ascitic fluid of DL-bearing mice (TAM); 1 x 10(5) cells activated with lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) and assayed for age-dependent alterations in macrophage tumoricidal functions such as: tumor cell binding, cytotoxicity, production of reactive nitrogen intermediates (RNI), expression of inducible nitric oxide synthase (iNOS) and oncostatin-M (OSM) were observed. TAM from old mice were observed to be inhibited with respect to tumor cell binding, cytotoxicity and expression of iNOS and OSM, as compared to macrophages of young and adult mice. TAM obtained from early tumor stages showed augmented tumor cytotoxicity as well as enhanced expression of iNOS and OSM in all the age groups. This effect was most pronounced in the TAM obtained from adult mice and least in the TAM obtained from old mice. The reasons for the observed difference are discussed. These observations should be helpful in understanding the effect of progressive tumor growth and age on the functions of TAM and their responsiveness towards therapeutic manipulations.

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.

LFA-1-dependent tumoricidal activity of cisplatin-treated macrophages.

The role of leucocyte function associated antigen-1 (LFA-1) (CD11a/18) in the tumoricidal activity of cisplatin-treated macrophages was investigated. Anti-LFA-1 antibodies inhibited cisplatin-induced macrophage cytotoxicity towards three different tumour cell lines. The decrease in tumoricidal activity of cisplatin-treated macrophages was attributed to their decreased binding to tumour cells in the presence of anti-LFA-1 (CD11a/18) antibodies. Western blot analysis revealed that cisplatin treatment leads to the expression of LFA-1 on macrophages which otherwise remains non-detectable. Because there is no information regarding the mechanism of cisplatin-induced LFA-1 expression and tumour cell binding by macrophages, the role of various second messenger molecules in these processes was investigated. Results suggest that protein phosphatase 2A (PP2A) is not involved in these processes whereas protein tyrosine phosphatases (PTP) negatively regulate LFA-1 expression and tumour-cell binding of cisplatin-treated macrophages. Inhibitors of protein phosphatase 1 (PP1), protein kinase C (PKC), protein tyrosine kinase (PTK), calmodulin and calmodulin-dependent kinase-II (CamK II) prevented LFA-1 expression on cisplatin-treated macrophages. A comparison with earlier results indicated that LFA-expression follows a distinct signalling pathway which is separate from the signalling pathway involved in NO or tumour necrosis factor/interleukin-1 (TNF/IL-1) expression in cisplatin-stimulated macrophages.

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.