Selective inhibition of glycoprotein and membrane protein of vesicular stomatitis virus from interferon-treated cells.

A 200-fold inhibition in the titer of infectious vesicular stomatitis virus (VSV) was produced in cultures of Ly cells treated with 30 reference units of interferon per milliliter. Virus particle production, as measured by VSV particle-associated transcriptase, or nucleocapsid protein was inhibited by a maximum of tenfold. The glycoprotein and membrane protein content was reduced in VSV derived from interferon-treated cells. Thus interferon-treated cells may have produced VSV particles with low infectivity, which may be related to the reduced amount of glycoprotein incorporated into such particles. These findings resemble those reported in interferon-treated cells infected with murine leukemia viruses.

Tunicamycin enhances the antiviral and anticellular activity of interferon.

The inhibitory effects of interferon on virus multiplication and cell growth are significantly enhanced by treatment with tunicamycin. Potentiation of antiviral activity was found only with enveloped viruses and not with nonbudding viruses. Changes in the plasma membrane of treated cells may account for this effect, since enveloped viruses bud from the cell surface as a terminal step.

Co-infection by Semliki forest virus and malarial parasite modulates viral multipucation, pathogenesis and cytokines in mice.

Environmental, technological and societal factors continue to have a dramatic effect on infectious diseases worldwide and are considered to be facilitating the emergence of several infectious diseases at a time. Co-infection with different species of viral and malaria infections are currently emerging problems of dual infection in the developing as well as developed countries. Understanding of interactions between the host, malaria and virus infection is of current concern and we have initiated studies to delineate the mechanisms involved during the progression of Semliki forest virus (SFV) and Plasmodium yoelii (P. yoelii) infection in mice. Enhanced virus multiplication and up-regulation of cytokine mRNA level in P. yoelii and SFV co-infected mice were observed on day 4 post-infection compared to respective controls. Collectively, our observations indicate that malaria infection may influence virus multiplication, pathogenesis and up-regulation of cytokine mRNA during co-infection in mice.

Phenotypic Detection and Antibiogram of ß-lactamase-producing Proteus Species in a Tertiary Care Hospital, India.

BACKGROUND: Proteus species cause a variety of community- and hospital-acquired illnesses. Synthesis of ß-lactamases is the predominant mechanism for resistance to ß-lactam antibiotics. Among the ß-lactamases, extended spectrum ß-lactamases (ESBLs) and AmpC ß-lactamases are the most common. AIM: The objective of this study was to determine the occurrence of ESBL and AmpC ß-lactamases in Proteus species among various clinical isolates at a tertiary care hospital, India. MATERIALS AND METHODS: This study was done to identify various species of Proteus from clinical samples (n = 3922). Antimicrobial susceptibility was performed by Kirby-Bauer disc diffusion method. ESBL production was detected by modified double-disc synergy test and indirect modified three-dimensional tests and AmpC ß-lactamase production by AmpC disc test and modified Hodge test. RESULTS: Proteus species were isolated in 5.4% (101/1876) specimens. Three Proteus species isolated were Proteus mirabilis 62.4% (63/101), Proteus vulgaris 29.7% (30/101), and Proteus penneri 7.9% (8/101). ESBL producers confirmed by both tests were of 88.1% (89/101). Only AmpC ß-lactamase was produced by four isolates. Coproduction of ESBL and AmpC ß-lactamase was observed in 58.4% (52/89) of isolates. Twelve isolates were non-ß-lactamase producers. Multidrug resistance (MDR) was found in 95.1% (96/101) of isolates, 50.5% (51/101) were possibly extensively drug resistant and none were pan drug resistant. None of the isolates were resistant to piperacillin-tazobactam. P. penneri isolates exhibited high resistance to most of the antibiotics. CONCLUSIONS: A high prevalence of ESBL and AmpC ß-lactamases was found that concurrently showed MDR. Phenotypic methods for the detection of ß-lactamases are easy and simple and can be implemented in routine diagnostic laboratories along with susceptibility testing. These data will assist the clinicians in the management and control of infections.

Thymosin beta4 accelerates wound healing.

Angiogenesis is an essential step in the repair process that occurs after injury. In this study, we investigated whether the angiogenic thymic peptide thymosin beta4 (Tbeta4) enhanced wound healing in a rat full thickness wound model. Addition of Tbeta4 topically or intraperitoneally increased reepithelialization by 42% over saline controls at 4 d and by as much as 61% at 7 d post-wounding. Treated wounds also contracted at least 11% more than controls by day 7. Increased collagen deposition and angiogenesis were observed in the treated wounds. We also found that Tbeta4 stimulated keratinocyte migration in the Boyden chamber assay. After 4-5 h, migration was stimulated 2-3-fold over migration with medium alone when as little as 10 pg of Tbeta4 was added to the assay. These results suggest that Tbeta4 is a potent wound healing factor with multiple activities that may be useful in the clinic.

Arnebin-1 accelerates normal and hydrocortisone-induced impaired wound healing.

Wound healing involves inflammation, cell proliferation, matrix deposition, and tissue remodeling. Interaction of different cells, extracellular matrix proteins, and their receptors are mediated by cytokines and growth factors during wound healing. In this study, we have evaluated the effect of arnebin-1, a natural product isolated from Arnebia nobilis, on normal and impaired wound healing in cutaneous punch wound model. Arnebin-1 was applied topically daily on wounds of hydrocortisone-treated or untreated animals. Arnebin-1 significantly accelerated healing of wounds with or without hydrocortisone treatment as revealed by a reduction in the wound width and gap length compared with controls. Arnebin-1 treatment promoted the cell proliferation, migration, and vessel formation to form a thick granulation tissue and re-epithelialization of the wounds. An increase in the synthesis of collagen, fibronectin and transforming growth factor-beta1 was seen in arnebin-1-treated wounds compared with the untreated control. As transforming growth factor-beta1 is known to enhance wound healing, and associated with the wound healing defect in hydrocortisone-treated wounds, the enhanced expression of transforming growth factor-beta1 at both translational and transcriptional level by arnebin-1 may be responsible for the enhancement of wound healing during normal and impaired wound repair. These studies suggest that arnebin-1 could be developed as a potent therapeutic agent for wound healing in steroid-impaired wounds.

Role of vacuolar H(+)-ATPase in interferon-induced inhibition of viral glycoprotein transport.

We have shown previously that interferon-beta (IFN-beta) induces the alkalinization of trans-Golgi network (TGN) and inhibits the transport of G protein of vesicular stomatitis virus (VSV) in L(B) cells and gD protein of herpes simplex virus (HSV-1) in LMtk- cells transfected with gD cDNA. The vacuolar H(+)-ATPase (V-ATPase) is responsible for maintaining pH in TGN, and V-ATPase-mediated acidification is required for normal transport of proteins. To examine whether alkalinization caused by IFN is mediated through V-ATPase, the activity of V-ATPase was determined in IFN-treated cells by coupling ATP hydrolysis to NADH oxidation. Bafilomycin (Baf) was used as positive control, as it specifically inhibits V-ATPase. The activity of V-ATPase was reduced in IFN-treated or Baf-treated cells compared with untreated cells. Doses of IFN-beta or Baf that neither alter pHi nor inhibit the transport of viral glycoproteins concomitantly inhibited the transport of G and gD proteins in TGN, as demonstrated by indirect immunofluorescence studies, and raised the pH of TGN as demonstrated by a decrease in the uptake of DAMP. Further, the effect of Baf on IFN-induced antiviral activity against VSV was examined to correlate the biologic significance of these findings. Data showed that Baf significantly enhances (5-50-fold) the IFN-induced antiviral activity as demonstrated by viral titers from supernatants. These findings suggest that the inhibition of transport of G and gD proteins by IFN-beta, may be related to the inhibition of V-ATPase-mediated acidification of TGN.

Killing of Leishmania donovani amastigotes by poly ICLC in hamsters.

In vitro as well as in vivo studies suggest that cytokine-induced synthesis of nitric oxide (NO) from L-arginine is a major effector mechanism against intracellular pathogens. In this study, we demonstrate that golden hamsters infected with Leishmania donovani amastigotes upon treatment with polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly ICLC), a potent interferon inducer and immune enhancer, in combination with L-arginine, develop the capacity to eliminate intracellular pathogens. This antileishmanial activity of poly ICLC was suppressed by N w nitro-L-arginine (N w NLA), an inhibitor of inducible NO synthase. Furthermore, prolonged treatment of infected animals with L-arginine alone for 5 days more after 5 day treatment with poly ICLC plus L-arginine increased the antileishmanial activity compared with 5 day treatment with poly ICLC plus L-arginine, suggesting that inducible NO synthase, once activated, produces NO for 5 days more. Our results suggest that an L-arginine-dependent, NO-mediated mechanism is probably responsible for the antileishmanial action of poly ICLC.

Mechanism of enhancement of the antiviral action of interferon against herpes simplex virus-1 by chloroquine.

Using double immunofluorescence, we have shown previously that interferon (IFN) treatment inhibits the transport of herpes simplex virus-1 (HSV-1) gD from the Golgi complex to the plasma membrane in the virus infected and gD cDNA transfected LMtk-cells. In the present study, we quantitated the gD protein on the cell surface and localized the gD protein in the trans-Golgi network (TGN). The results showed 10-fold less fluorescence for the gD protein on the cell surface in IFN-treated LMtk-cells. Subcellular fractionation studies demonstrated that gD was associated with TGN-enriched membranes. Gold labeling for DAMP distribution using electron microscopy showed that IFN raised the pH of TGN. IFNs induced alkalinization of TGN may be related to the block in the transport of HSV-1 gD. Earlier we reported that a subeffective dose of chloroquine (CHL) or IFN does not change the pHi. However, both CHL and IFN together raise the pHi significantly. To study the biologic significance of the finding, the effect of these subeffective doses of IFN and CHL on the antiviral activity and the transport of the gD protein was studied. Results suggested that CHL enhance the antiviral activity of IFN against HSV-1 and concomitantly increase the inhibition of HSV-1 gD transport. This IFN-induced increase in pHi of the TGN may also explain the inhibitory effect of IFN reported on the terminal steps of some of the enveloped viruses.

Poly ICLC inhibits Plasmodium cynomolgi B malaria infection in rhesus monkeys.

Prophylatic treatment with a single dose of 1.0 or 2.0 mg/kg (body weight) of polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (Poly ICLC), a potent interferon (IFN) inducer and immune enhancer, 18 h before intravenous inoculation of sporozoites (1.04 x 10(5)-0.70 x 10(6) sporozoites) of Plasmodium cynomolgi B in the rhesus monkey, completely abolished the infectivity of sporozoites. The inhibitory effect of Poly ICLC is dose dependent in monkeys infected with P. cynomolgi B sporozoites. Treatment with lower doses of Poly ICLC (0.5 mg/kg) provided significant protection, but the lowest dose of Poly ICLC used (0.1 mg/kg) failed to provide any protection. Prophylactic treatment with Poly ICLC, however, had no protective effect against trophozoite-induced infection.

Immunochemotherapy for Leishmania donovani infection in golden hamsters: combinatorial action of poly ICLC plus L-arginine and sodium stibogluconate (Stibanate).

We demonstrate that golden hamsters infected with Leishmania donovani amastigotes develop the capacity to eliminate intracellular pathogens on treatment with low-dose standard antileishmanial sodium stibogluconate (Stibanate) in combination with polyinosinic-polycytidilic acid stabilized with polylysine and carboxymethycellulose (poly ICLC), a potent inducer of interferon (IFN) and immune enhancer, plus L-arginine. Data suggest that low doses of both Stibanate and poly ICLC plus L-arginine provide marginal inhibition against L. donovani infection in golden hamsters. When given in combination, however, a significant inhibition was achieved without toxicity, as all the animals survived up to 45 or 60 days. These results suggest that combination therapy using Stibanate and poly ICLC plus L-arginine may be very effective in reducing the dose of Stibanate and, hence, its dose-dependent toxicity in clinical situations.

Poly ICLC enhances the antimalarial activity of chloroquine against multidrug-resistant Plasmodium yoelii nigeriensis in mice.

Swiss mice infected with multidrug-resistant Plasmodium yoelii nigeriensis were treated with polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethyl cellulose (Poly ICLC), a potent interferon (IFN) inducer and immune enhancer, in combination with chloroquine (CQ), which completely eliminated the malaria parasite from these animals. The enhancement of the antimalarial activity of poly ICLC was found to be completely reversed by the cytochrome P-450 inducer, phenobarbitone. No effect of Nw nitro-L-arginine (NLA), an inhibitor of nitric oxide, was seen on the enhancement of the antimalarial activity of CQ by Poly ICLC. These results suggest the possible involvement of cytochrome P-450 enzyme-mediated mechanism in the enhancement of the antimalarial activity of CQ by Poly ICLC.

Thrombin stimulation of synthesis and secretion of fibronectin by human A549 epithelial cells and mouse LB fibroblasts.

Thrombin, a serine protease generated at wound sites, takes part in multiple biological functions, including wound healing. The present report elucidates the effect of thrombin on fibronectin (FN) synthesis and secretion in fibroblasts and epithelial cells. Subconfluent cultures of mouse LB fibroblasts and human A549 epithelial cells were exposed to various concentrations of bovine plasma thrombin at 37 degrees C for 16 hr. After exposure, cells were processed for determination of cell-associated and secreted FN by metabolic labeling, immunoprecipitation, immunofluorescence, and peroxidase immunocytochemistry. The correlation of FN production with cell growth was studied by a combined procedure of peroxidase immunocytochemistry and light microscopic autoradiography. The amounts of cell-associated and secreted FN were significantly increased with dose increments of thrombin. The increases were most evident in secreted FN. The increase of cell-associated FN was also evidenced by results from immunofluorescence and immunocytochemical studies. Ultrastructurally, the intracellular FN was localized in rough endoplasmic reticulum, Golgi complexes, and secretory granules, whereas non-released extracellular FN was localized in the plasma membrane of cell-to-cell contacts and in the extracellular fibrils. More intense cytoplasmic FN staining was observed in cells that were not labeled with [3H]-thymidine, indicating that FN production may vary with different phases of cell growth. The results imply that thrombin may play an important role in the early phases of wound healing.

Brefeldin A inhibits the antiviral action of interferon against encephalomyocarditis virus.

Brefeldin A (BFA), a unique fungal metabolite of a 13-membered lactone ring, exhibits various biological actions, including antitumor, antifungal and antiviral activities. In the present study, mouse LB cells were treated with various concentrations of interferon (IFN) and/or BFA overnight and infected with encephalomyocarditis virus (EMCV) after removal of IFN and BFA. Doses of BFA which neither inhibit the metabolism of the cell nor the infectivity of EMCV, decreased the IFN-induced antiviral activity against EMCV as demonstrated by virus titer from supernatants. Since 2-5A synthetase and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) have been suggested to be involved in the antiviral action of IFN against EMCV, their activities were investigated in LB cells after BFA treatment. Northern blot analysis and in situ hybridization showed a decrease (2-3-fold) in the mRNA of 2'-5' oligoadenylate (2-5A) synthetase after BFA treatment. BFA also inhibited the activity of 2-5A synthetase, 2-5A dependent RNase and phosphorylation of PKR in cellular extracts, indicating that BFA may be exerting its inhibitory effect both at the transcriptional and post-transcriptional levels. This study reports a new biological action of BFA, demonstrating that BFA antagonized the antiviral action of IFN by inhibiting IFN-induced enzymatic pathways. These studies also suggest that both 2-5A and PKR are important in the antiviral activity of IFN against EMCV.

Effects of interferon in malaria infection.

Earlier, we reported that prophylactic treatment with human interferon gamma (rHuIFN-gamma) protected monkeys against Plasmodium cynomolgi B malaria infection. We have tested the efficacy of rHuIFN-gamma on relapsing stage of experimental P. cynomolgi B malaria infection in rhesus monkeys. No effect of rHuIFN-gamma was seen against experimental relapsing stage compared with controls; however, it appears that chloroquine (CHL) may have interfered with the antimalarial effect of IFN, since treatment with CHL inhibits the antiviral activity of mouse alpha/beta IFN and polyinosinic-polycytidylic acid (poly I:C) against Semliki forest virus (SFV) in mice. These results may have clinical implications especially with the use of IFN against virus infection, cancer and in parasitic infections in malaria endemic areas where CHL is one of the most widely used antimalarial drugs. Our result also shows that CHL treatment enhances the virus replication in mice and suggest a possible connection between AIDS and malaria infection, since the spread of AIDS has been rapid in parts of tropical Africa that have a high incidence of malaria, and chloroquine has been frequently used in the chemotherapy of malaria.

Curcumin inhibits the proliferation and cell cycle progression of human umbilical vein endothelial cell.

We have studied the effect of curcumin (diferuloylmethane), a major component of the food flavor turmeric, on the proliferation and cell cycle progression of human umbilical vein endothelial cells (HUVEC). Curcumin inhibited the DNA synthesis of HUVEC as revealed by [3H]thymidine incorporation in a dose-dependent manner without significantly affecting the viability of the cells. The growth of HUVEC stimulated with fibroblast growth factor (FGF) and endothelial growth supplement (ECGS) was also inhibited by curcumin. Addition of curcumin to HUVEC resulted in an accumulation of > 46% of the cells in early S-phase, as determined by the FACS analysis. Pulse labeling studies with [3H]thymidine demonstrated that curcumin affected cells that were actively undergoing DNA synthesis. The de-novo synthesis of thymidine depends on thymidine kinase (TK) enzyme. Curcumin caused a significant loss of TK activity, which may be one of the possible mechanism(s) for the inhibition of DNA synthesis activity of HUVEC by curcumin. These studies have revealed a unique mode of action of curcumin whereby it effectively blocked the cell cycle progression during S-phase by inhibiting the activity of TK enzyme. The migration, proliferation and differentiation of HUVEC leads to angiogenesis, which facilitates the tumor initiation and promotion. Since curcumin inhibited the proliferation of HUVEC, it could turn out to be a very useful compound for the development of novel anti-cancer therapy.

Prevention of renal ischemia-reperfusion-induced injury in rats by picroliv.

Picroliv is a potent antioxidant extracted from the roots and rhizome of Picrorhiza kurrooa. It has been shown to impart significant hepatoprotective activities, partly by modulation of free radical-induced lipid peroxidation. Lipid peroxidation and reactive oxygen species are associated with tissue injury in post-ischemic acute renal failure. The efficacy of picroliv was assessed in an in vivo model of renal ischemia-reperfusion injury (IRI) in rats at a dose of 12 mg/kg orally for 7 days. The animals were killed at various times after reperfusion. Increased lipid peroxidation and apoptotic cell number reflected the oxidative damage following renal IRI. Picroliv-pretreated rats exhibited lower lipid peroxidation, improved antioxidant status, and reduced apoptosis, indicating better viability of renal cells. Immunohistochemical studies revealed that picroliv pretreatment attenuated the expression of intercellular adhesion molecule-1 in the glomerular region. These results suggested that picroliv pretreatment protects rat kidneys from IRI, perhaps by modulation of free radical damage and adhesion molecules.

Acceleration of viral replication and up-regulation of cytokine levels by antimalarials: implications in malaria-endemic areas.

Antimalarial drugs are widely used in malaria endemic areas, both for chemoprophylaxis and also empirically to treat patients presenting with fever. Previously, we have reported that chloroquine enhances the severity of Semliki forest virus (SFV) and encephalomyocarditis virus infection. The studies presented herein show that a broad spectrum of antimalarial drugs augmented the replication of SFV in mice, concomitant with greater tissue damage and up-regulation of mRNA levels of various inflammatory cytokine genes, including interleukin-1 receptor antagonist (IL-1Ra), II-1alpha, IL-1beta, IL-6, IL-12p40, and interferon-gamma inducing factor. Furthermore, chloroquine enhances IL-1Ra production in RAW cells in vitro. Since IL-1Ra is known to be up-regulated in a number of viral infections, we propose that a further enhancement of its expression by antimalarials may be responsible for the increased severity of viral infection in our studies. Thus, the widespread use of antimalarials in malaria-endemic areas may predispose the population to viral infections. Further studies are in progress to delineate mechanism(s) involved in cytokine up-regulation and acceleration of viral replication.

Picroliv preconditioning protects the rat liver against ischemia-reperfusion injury.

Cell death following ischemia-reperfusion injury is a major concern in clinical issues such as organ transplantation and trauma. The need to identify agents with a potential for preventing such damage has assumed great importance. We have evaluated the efficacy of picroliv, a potent antioxidant derived from the plant Picrorhiza kurrooa, in protecting against hepatic ischemia-reperfusion injury in vivo. Picroliv was fed to male Sprague Dawley rats in a dose of 12 mg/kg once daily by oral gavage for 7 days prior to hepatic ischemia. Ischemia was induced by occluding the hepatic pedicel with a microaneurysm clip for 30 min and reperfusion was allowed thereafter for varying period (15-120 min) by releasing the microaneurysm clip. Picroliv pretreatment resulted in better hepatocyte glycogen preservation and reduced apoptosis. Reduction in apoptosis was associated with decreased mRNA expression of caspase-3 and Fas. Oxidant induced cellular damage as measured by tissue malondialdehyde (MDA) levels was significantly less following picroliv pretreatment. Both a reduction in neutrophil infiltration and an increased level of intracellular antioxidant enzyme superoxide dismutase possibly contributed to the reduction in tissue lipid peroxidation. Tissue inflammatory cytokines level of interleukin-1alpha (IL-1alpha) and interleukin-1beta (IL-1beta) was also lower in picroliv group. Furthermore, picroliv pretreatment resulted in enhanced proliferating cell nuclear antigen (PCNA) immunoreactivity. These studies strongly suggest picroliv to be a promising agent for ameliorating injury following ischemia-reperfusion.

Inhibition of growth and regulation of IGFs and VEGF in human prostate cancer cell lines by shikonin analogue 93/637 (SA).

BACKGROUND: Insulin-like growth factors (IGFs) are important mitogens and are involved in normal and malignant cellular proliferation. IGFs and IGF binding proteins (IGFBPs) regulate the prostatic cell growth and reduction/blocking of IGFs has been suggested to be of therapeutic value in prostate cancer. beta,beta-dimethyl acryl shikonin, an extract from the roots of plant Arnebia nobilis has been shown to have anticancer properties but was found to be toxic. Subsequently, several analogoues of beta,beta-dimethyl acryloyl shikonin were synthesized and one of them shikonin analogue 93/637 (SA) was significantly less toxic compared to beta,beta-dimethyl acryloyl shikonin. MATERIALS AND METHODS: We have investigated the effect of SA on prostate cancer cell (DU 145, LNCaP and PC-3) growth and expression of IGFs (IGF-I, IGF-II and IGF-I receptor (IGF-IR)), IGFBP-3 and vascular endothelial growth factor (VEGF). RESULTS: SA had growth inhibitory effect on PC-3 cells in a dose dependent manner. It also showed slight inhibitory effect on the growth of DU 145 and LNCaP cells at low doses ranging from 250 nM to 1 microM and has moderate inhibitory effect at concentrations 2.5 microM and above. Lactate dehydrogenase (LDH) activity assays indicated cellular damage, only at higher concentrations of SA that are greater than 1 microM. Gene expression studies by RT-PCR have demonstrated a decrease in mRNAs of IGF-II in DU 145, IGF-I, and IGF-IR in LNCaP, and IGF-II and VEGF in PC-3 cells and an increase in IGFBP-3 in both DU 145 and PC-3 cells by treatment with SA. CONCLUSIONS: The results demonstrate the inhibitory effect of SA on cellular growth and IGFs specifically in PC-3 cells and suggest a potential therapeutic use in treatment of prostate cancer.