VCAM-1 expression on CD8+ cells correlates with enhanced anti-HIV suppressing activity.

CD8(+) cells from HIV-infected individuals showing the CD8(+) cell noncytotoxic antiviral response unexpectedly revealed mRNA for VCAM-1, a cell surface molecule found on endothelial cells. Uninfected subjects had undetectable levels of VCAM-1 mRNA in their CD8(+) cells. Flow cytometry analysis showed that up to 12% of the CD8(+) cells from HIV-positive individuals expressed VCAM-1 compared with 0.8% of the CD8(+) cells of HIV-negative individuals. Enrichment of the CD8(+)VCAM-1(+) cell population and subsequent coculture with CD4(+) cells acutely infected with HIV-1 showed that the VCAM-1(+)CD8(+) cells were able to suppress viral replication with 50% less input cells than the unseparated CD8(+) cell population. This study demonstrates, for the first time, the expression of VCAM-1 on CD8(+) cells. Moreover, the CD8(+)VCAM-1(+) cells show enhanced CD8(+) cell noncytotoxic antiviral response activity that could have clinical importance in HIV infection.

Genetic manipulation of telomerase in HIV-specific CD8+ T cells: enhanced antiviral functions accompany the increased proliferative potential and telomere length stabilization.

A large proportion of the CD8(+) T cell pool in persons chronically infected with HIV consists of cells that show features of replicative senescence, an end stage characterized by irreversible cell cycle arrest, multiple genetic and functional changes, and shortened telomeres. The objective of our research was to determine whether constitutive expression of the gene for the human telomerase (hTERT) can prevent senescence-induced impairments in human virus-specific CD8(+) T cells, particularly in the context of HIV-1 disease. Our results indicate that hTERT-expressing HIV-specific CD8(+) lymphocytes show both an enhanced and sustained capacity to inhibit HIV-1 replication in in vitro coculture experiments, as well as prolonged ability to produce IFN-gamma and TNF-alpha in response to stimulation with HIV-1-derived peptides, as compared with vector-transduced controls. Loss of CD28 expression, the signature change of replicative senescence in cell culture, was retarded in those CD8(+) T cell cultures that had high levels of CD28 at the time of hTERT transduction. These findings suggest that telomere shortening may be the primary driving force behind several aspects of CD8(+) T cell dysfunction associated with replicative senescence. We also demonstrate reduced accumulation of the p16(INK4a) and p21(WAF1) cell cycle inhibitors in hTERT-transduced lymphocytes, providing a possible mechanism by which stable hTERT expression is able to circumvent the senescence barrier in CD8(+) T cells. Given the key role of CD8(+) T cell function in controlling a variety of acute and latent viral infections, approaches to retard the functional decrements associated with replicative senescence may lead to novel types of immunotherapy.

A nonneutralizing anti-HIV-1 antibody turns into a neutralizing antibody when expressed on the surface of HIV-1-susceptible cells: a new way to fight HIV.

During HIV-1 infection or vaccination, HIV-1 envelope spikes elicit Ab responses. Neutralizing Abs block viral entry by recognizing epitopes on spikes critical for their interaction with receptor, coreceptors or fusion. In contrast, nonneutralizing Abs fail to do so because they recognize epitopes either buried or exposed but not critical for viral entry. Previously, we produced a high-affinity human mAb against the cluster II determinant of gp41. This Ab or its recombinant Fab and single-chain Fv have been repeatedly shown to bind to HIV-1 gp160 or gp41, but fail to block viral entry. We report that, surprisingly, expression of this nonneutralizing anti-HIV-1 gp41 single-chain Fv on the surface of human CD4 T cells markedly inhibits HIV-1 replication and cell-cell fusion. The inhibition targets the HIV-1 envelope at the level of viral entry, regardless of HIV-1 tropism. Although this bona fide nonneutralizing Ab does not neutralize HIV-1 entry when produced as a soluble protein, it acts as a neutralizing Ab when expressed on the cell surface. Expressing Abs on the surface of HIV-1-susceptible cells can be a new way to fight HIV-1.

Genomic effects of IFN-beta in multiple sclerosis patients.

The purpose of this report was to characterize the dynamics of the gene expression cascades induced by an IFN-beta-1a treatment regimen in multiple sclerosis patients and to examine the molecular mechanisms potentially capable of causing heterogeneity in response to therapy. In this open-label pharmacodynamic study design, peripheral blood was obtained from eight relapsing-remitting multiple sclerosis patients just before and at 1, 2, 4, 8, 24, 48, 120, and 168 h after i.m. injection of 30 micro g of IFN-beta-1a. The total RNA was isolated from monocyte-depleted PBL and analyzed using cDNA microarrays containing probes for >4000 known genes. IFN-beta-1a treatment resulted in selective, time-dependent effects on multiple genes. The mRNAs for genes implicated in the anti-viral response, e.g., double-stranded RNA-dependent protein kinase, myxovirus resistance proteins 1 and 2, and guanylate binding proteins 1 and 2 were rapidly induced within 1-4 h of IFN-beta treatment. The mRNAs for several genes involved in IFN-beta signaling, such as IFN-alpha/beta receptor-2 and Stat1, were also increased. The mRNAs for lymphocyte activation markers, such as IFN-induced transmembrane protein 1 (9-27), IFN-induced transmembrane protein 2 (1-8D), beta(2)-microglobulin, and CD69, were also increased in a time-dependent manner. The findings demonstrate that IFN-beta treatment induces specific and time-dependent changes in multiple mRNAs in lymphocytes of multiple sclerosis patients that could provide a framework for rapid monitoring of the response to therapy.

A soluble chemokine-binding protein from vaccinia virus reduces virus virulence and the inflammatory response to infection.

Many poxviruses express a secreted protein that binds CC chemokines with high affinity and has been called viral CC chemokine inhibitor (vCCI). This protein is unrelated to any known cellular protein, yet can compete with host cellular CC chemokine receptors to modulate host inflammatory and immune responses. Although several strains of vaccinia virus (VV) express a vCCI, the best characterized VV strains Western Reserve and Copenhagen do not. In this study, we have expressed the vCCI from VV strain Lister in a recombinant Western Reserve virus (v Delta B8R-35K) and characterized its binding properties in vitro and its effect on virulence in vivo relative to wild-type virus (v Delta B8R) or a revertant virus (v Delta B8R-R) where Lister 35-kDa had been removed. Cells infected with v Delta B8R-35K secreted a 35-kDa protein that bound the CC chemokine macrophage-inflammatory protein 1 alpha. Expression of vCCI attenuated the virus in a murine intranasal model, characterized by reduced mortality and weight loss, decreased virus replication and spread, and a reduced recruitment of inflammatory cells into the lungs of VV-infected mice. The CC chemokines macrophage-inflammatory protein 1 alpha, eotaxin, and macrophage chemotactic protein 1 were detected in bronchoalveolar lavage fluids from v Delta B8R-infected mice; however, bronchoalveolar lavage fluids from v Delta B8R-35K-infected mice had lower levels of chemokines and a reduced chemotactic activity for murine leukocytes in vitro. These observations suggest that vCCI plays an important role in regulating leukocyte trafficking to the lungs during VV infection by binding to CC chemokines and blocking their chemotactic activities.

An antiviral state induced in Chinook salmon embryo cells (CHSE-214) by transfection with the double-stranded RNA poly I:C.

Type I interferons (IFN alpha and beta) convert vertebrate cells into an antiviral state by inducing expression of proteins that inhibit virus replication. In humans and mice, Mx proteins constitute one family of interferon-induced antiviral proteins. Mx genes have recently been cloned from Atlantic salmon and rainbow trout. Moreover, double-stranded RNA (dsRNA) and type I IFN-like activity have been shown to induce Mx protein in salmonid cells. Chinook salmon embryo cells (CHSE-214 cells) have been suggested to have a defect in the IFN-system because the dsRNA polyinosinic polycytidylic acid (poly I:C) failed to induce an antiviral state in the cells. We have studied this phenomenon more closely in the present work. CHSE-214 cells were either transfected with poly I:C or incubated with poly I:C without transfection reagent. The cells were then studied for Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) infection. The results showed that cells transfected with poly I:C were protected from IPNV infection, whilst cells incubated with poly I:C were not protected. Cells transfected with the double-stranded DNA poly dI:dC were also not protected against IPNV. Mx protein was expressed in CHSE-214 cells upon transfection with poly I:C, but not after incubation with poly I:C alone. Stimulation of CHSE-214 cells with supernatants from cells transfected with poly I:C, induced protection against IPNV, indicating production of type I IFN-like activity. These results suggest that CHSE-214 cells in fact are able to produce type I IFN, but may have defects in the mechanisms mediating uptake of poly I:C or may degrade unprotected poly I:C.

Effect of poly I:C on the expression of Mx proteins and resistance against infection by infectious salmon anaemia virus in Atlantic salmon.

Mx proteins are induced by type I interferons (IFN alpha and beta) in mice and humans and inhibit the replication of orthomyxoviruses and some other single-stranded RNA viruses. Recently, Mx genes have been cloned from Atlantic salmon. Mx transcripts were shown to be induced in head-kidney, liver and gills of the fish by the synthetic double-stranded RNA polyinosinic polycytidylic acid (poly I:C). In the present work we have studied expression of Mx protein in organs of Atlantic salmon treated with poly I:C. A quantitative immunoblot method was established to monitor expression of Mx protein and to compare relative amounts of Mx protein in different organs. Treatment of Atlantic salmon with poly I:C increased the relative amount of Mx protein in liver, stomach, hindgut, head-kidney and spleen. In gills the levels of Mx protein were similar in control fish and poly I:C treated fish. Immunohistochemistry of tissue sections from liver, head-kidney and gills from poly I:C treated fish was in accordance with the immunoblotting data and showed staining for Mx protein in several different cell types. Classification of infectious salmon anaemia virus as an orthomyxovirus makes it a putative target for Atlantic salmon Mx protein. Atlantic salmon treated with poly I:C showed reduced cumulative mortality compared to the control fish when challenged with infectious salmon anaemia virus (ISAV) by intraperitoneal injection. This demonstrates that poly I:C has some protective effect against ISAV in vivo.

Effect of double-stranded RNA and interferon on the antiviral activity of Atlantic salmon cells against infectious salmon anemia virus and infectious pancreatic necrosis virus.

Type I interferons (IFN) establish an antiviral state in vertebrate cells by inducing expression of Mx and other antiviral proteins. We have studied the effect of Atlantic salmon interferon-like activity (AS-IFN) and poly I:C on the Mx protein expression and antiviral activity against infectious salmon anaemia virus (ISAV) and infectious pancreatic necrosis virus (IPNV) in the Atlantic salmon cell lines SHK-1 and TO. The double-stranded RNA poly I:C is an inducer of type I IFN in vertebrates. A cell cytotoxicity assay and measurements of virus yield were used to measure protection of cells against virus infection. Maximal induction of Mx protein in TO and SHK-1 cells occurred 48 h after poly I:C stimulation and 24 h after AS-IFN stimulation. TO cells pretreated with AS-IFN or poly I:C were protected from infection with IPNV 24 to 96 h after stimulation. Poly I:C or AS-IFN induced a minor protection against ISAV infection in SHK-1 cells, but no protection was induced against ISAV in TO cells. Western blot analysis showed that ISAV induced expression of Mx protein in TO and SHK-1 cells whereas IPNV did not induce Mx protein expression. These results suggest that ISAV and IPNV have very different sensitivities to IFN-induced antiviral activity and have developed different strategies to avoid the IFN-system of Atlantic salmon. Moreover, Atlantic salmon Mx protein appears not to inhibit replication of ISAV.

A comparative investigation of CC chemokines and SIV suppressor factors generated by CD8+ and CD4+ T cells and CD14+ monocytes.

The capacity of CD8+ and CD4+ T cells and CD14+ monocytes to generate the CC chemokines, RANTES, MIP-1alpha and MIP-1beta, and SIV suppressor factors were studied using cells separated from PBMC of macaques immunized with the 70-kDa heat shock protein (HSP70). Unimmunized macaques showed low levels of the three CC chemokines and SIV-SF, and they showed little variation between PBMC and the two subsets of T cells stimulated with PHA. Immunization with HSP70 elicited an increase in the in vitro concentration of each of the three CC chemokines and SF. This was found with PBMC, CD4+ and CD8+ T cells and to a lesser extent with monocytes, when conventionally separated enriched cell subsets were examined from the same PBMC. However, the concentrations of the three CC chemokines derived from highly purified cell-sorted populations (>95%) were greatly increased, as compared with the enriched cell subsets. The concentration of each of the three chemokines was highest for CD8+ T cells, decreased with CD4+ T cells and was lowest with the CD14+ monocytes, but the latter were not stimulated. Neutralization assays with antibodies to the three CC chemokines showed that the antiviral activity generated by the four populations of cells could be largely accounted for by the three CC chemokines. The results of this comparative study suggests that CD8+ as well as CD4+ T cells and CD14+ monocytes generate the three CC chemokines and SIV-SF when stimulated with a mitogen, and that the baseline innate level can be upregulated by adaptive immune responses to a specific antigen.

Increased levels of antiviral MxA protein in peripheral blood of patients with a chronic disease of unknown etiology.

Interferon alpha (IFN-alpha) is synthesized in response to viral infections. MxA protein, induced specifically by IFN-alpha and beta, expressed in peripheral blood cells, is detected more consistently than circulating IFN-alpha in serum of patients with viral infections. Thus, activation of the IFN-alpha/MxA system can be used as additional marker of the presence of a virus in patients. Therefore MxA protein and IFN-alpha levels were measured in patients with multiple sclerosis (MS), a chronic neurological disease of unknown etiology, in order to investigate the possible role of viruses in the expression of this disease. The means of MxA values obtained by using an immunochemiluminescent assay were significantly higher in blood of patients with remitting (n = 197) or relapsing (n = 39) multiple sclerosis (MS) patients and in patients with viral infections than in blood from healthy controls (n = 25) and from patients with bacterial infections (n = 12). Intra-individual variance in MxA levels in seven clinically stable remitting patients with MS was observed in the course of a follow-up, and high MxA levels were detected in three of them in blood samples collected consecutively over several months. By using an ultra sensitive assay, a higher MxA-inducer activity was obtained with sera from MS patients (n = 39) than with those from healthy controls (n = 12). Experiments with neutralizing antibodies proved that this activity in serum from patients was due to IFN-alpha, whereas IFN-alpha could not be detected by other methods. Altogether these results demonstrate that there is an activation of the IFN-alpha/MxA system in MS patients, which is consistent with the hypothesis that a viral infection may be associated with MS.

Caveolin-1 expression is down-regulated in cells transformed by the human papilloma virus in a p53-dependent manner. Replacement of caveolin-1 expression suppresses HPV-mediated cell transformation.

Squamous cell carcinomas of the lung and cervix arise by neoplastic transformation of their respective tissue epithelia. In the case of cervical carcinomas, an increasing body of evidence implicates the human papillomavirus, HPV (types 16 and 18), as playing a pivotal role in this malignant transformation process. The HPV early genes E6 and E7 are known to inactivate the tumor suppressors p53 and Rb, respectively; this leads to disruption of cell cycle regulation, predisposing cells to a cancerous phenotype. However, the role of caveolin-1 (a putative tumor suppressor) in this process remains unknown. Here, we show that caveolin-1 protein expression is consistently reduced in a panel of lung and cervical cancer derived cell lines and that this reduction is not due to hyperactivation of p42/44 MAP kinase (a known negative regulator of caveolin-1 transcription). Instead, we provide evidence that this down-regulation event is due to expression of the HPV E6 viral oncoprotein, as stable expression of E6 in NIH 3T3 cells is sufficient to dramatically reduce caveolin-1 protein levels. Furthermore, we demonstrate that p53-a tumor suppressor inactivated by E6-is a positive regulator of caveolin-1 gene transcription and protein expression. SiHa cells are derived from a human cervical squamous carcinoma, harbor a fully integrated copy of the HPV 16 genome (including E6), and show dramatically reduced levels of caveolin-1 expression. We show here that adenoviral-mediated gene transfer of the caveolin-1 cDNA to SiHa cells restores caveolin-1 protein expression and abrogates their anchorage-independent growth in soft agar. Taken together, our results suggest that the HPV oncoprotein E6 down-regulates caveolin-1 via inactivation of p53 and that replacement of caveolin-1 expression can partially revert HPV-mediated cell transformation.

IL-1 beta attenuates IFN-alpha beta-induced antiviral activity and STAT1 activation in the liver: involvement of proteasome-dependent pathway.

IFN-alphabeta is the only established treatment for viral hepatitis; however, more than 60% of patients are poorly responsive. Because viral hepatitis is associated with inflammation, we hypothesized that inflammation may attenuate the efficacy of IFN therapy. To test this hypothesis, the effect of IL-1beta, one of the major proinflammatory cytokines, on IFN signaling pathway in the liver was examined. Administration of IL-1beta in vivo attenuated IFN-alphabeta-induced STAT1 tyrosine phosphorylation in the liver but not in the spleen. The inhibitory action of IL-1beta in vivo was not affected by depleting hepatic Kupffer cells, suggesting that IL-1beta may directly target IFN-alphabeta signaling in hepatocytes. Indeed, pretreatment of human hepatocellular carcinoma HepG2 cells with IL-1beta suppressed IFN-alphabeta-induced antiviral activity and antiviral protein MxA mRNA expression. Furthermore, IL-1beta attenuated IFN-alphabeta-induced STAT1 binding and tyrosine phosphorylation without affecting the level of STAT1 protein. This inhibitory effect can be reversed by pretreatment with either proteasome inhibitors or transfection of dominant negative NF-kappaB inducing kinase mutants. Taken together, these findings suggest that IL-1beta attenuates IFN-alphabeta-induced STAT1 activation by a proteasome-dependent mechanism. In view of high levels of IL-1beta in the serum or within the liver of patients with chronic liver diseases, attenuation of IFN-alphabeta signaling in the liver by IL-1beta could be one of the mechanisms underlying the resistance to IFN therapy in chronic hepatitis C, and IL-1beta could be a potential therapeutic target for improving the efficacy of IFN therapy.

Induction of Mx protein by interferon and double-stranded RNA in salmonid cells.

Mx protein is one of several antiviral proteins that are induced by the type I interferons (IFN), IFNalpha and beta, in mammals. In this work induction of a 76 kDa Mx protein by double-stranded RNA (dsRNA) or type I IFN-like activity in Atlantic salmon macrophages, Atlantic salmon fibroblast cells (AS cells) and in Chinook salmon embryo cells (CHSE-214) is reported. Type I IFN-like activity was produced by the stimulation of Atlantic salmon macrophages with the synthetic dsRNA polyinosinic polycytidylic acid (poly I:C). A correlation appeared to exist between Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) induced by IFN in CHSE-214 cells. Several observations in the present work suggest that, as in mammals, the induction of Mx protein by dsRNA in fish cells primarily occurs via induction of type I IFN. First, type I IFN-like activity but not poly I:C, induced Mx protein expression in CHSE-214 cells. These cells apparently lack the ability to produce IFN in response to poly I:C. Second, the putative IFN induced maximal Mx protein expression 48 h earlier than poly I:C in AS cells. Third, the peak expression of Mx protein in macrophages induced by poly I:C occurred after 48 h whereas peak in IFN-like activity was observed by 24 h after addition of poly I:C. The present work supports the notion of using Mx protein as a molecular marker for the production of putative type I IFN in fish.

Activation of cellular interferon-responsive genes after infection of human cells with herpes simplex virus type 1.

Previous studies have shown that infection of human fibroblasts with human cytomegalovirus (HCMV) results in activation of cellular interferon-responsive gene expression. We demonstrate here that infection of human fibroblasts with herpes simplex virus type 1 (HSV-1) in the absence of de novo protein synthesis also induces the expression of interferon-responsive genes. Five genes tested (encoding ISG54, IFI56, ISG15, 9-27 and MxA) were activated by infection with HSV-1, although the degree of response varied between the individual genes. HSV-1 was a less efficient inducer than HCMV. The effect was a consequence of binding of the virus particle to the cell surface or of the presence of virion components within the infected cell. Induction was mediated by a pathway other than the mechanism through which interferon-alpha mediates its effects on cellular gene expression.

Mx mRNA expression and RFLP analysis of rainbow trout Oncorhynchus mykiss genetic crosses selected for susceptibility or resistance to IHNV.

Three interferon-inducible Mx genes have been identified in rainbow trout Oncorhynchus mykiss and their roles in virus resistance have yet to be determined. In mice, expression of the Mx1 protein is associated with resistance to influenza virus. We report a study to determine whether there was a correlation between the expression of Mx in rainbow trout and resistance to a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A comparison of Mx mRNA expression was made between different families of cultured rainbow trout selected for resistance or for susceptibility to IHNV. A trout-specific Mx cDNA gene probe was used to determine whether there was a correlation between Mx mRNA expression and resistance to the lethal effects of IHNV infection. Approximately 99% of trout injected with a highly virulent strain of the fish rhabdovirus, IHNV, were able to express full length Mx mRNA at 48 h post infection. This is markedly different from the expression of truncated, non-functional Mx mRNA found in most laboratory strains of mice, and the ability of only 25% of wild mice to express functional Mx protein. A restriction fragment length polymorphism (RFLP) assay was developed to compare the Mx locus between individual fish and between rainbow trout genetic crosses bred for IHNV resistance or susceptibility. The assay was able to discriminate 7 distinct RFLP patterns in the rainbow trout crosses. One cross was identified that showed a correlation between homozygosity at the Mx locus and greater susceptibility to IHN-caused mortality.

Antiviral effect of nitric oxide during Japanese encephalitis virus infection.

The ability of Japanese encephalitis virus (JEV) and JEV-induced macrophage derived neutrophil chemotactic factor (MDF) to produce nitric oxide (NO), and the possible antiviral effect of NO during JEV infection, was investigated. Splenic macrophages of JEV infected mice produced maximum NO in vivo at day 7 post infection, and in vitro at 24 h after JEV stimulation. MDF-induced NO production was dose dependent and maximal at 60 min after MDF treatment. The response was sensitive to anti-MDF antibody treatment and the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). Pretreatment of mice with L-NMMA increased the mortality to 100% in JEV infected mice in vivo and inhibited NO production in vitro, while MDF stimulated macrophages inhibited virus replication with high levels of NO production. MDF treatment increased the survival rate of JEV infected mice. The findings thus demonstrate that MDF induces production of NO during JEV infection, which has an antiviral effect. This may be one of the important mechanisms of natural immunity in controlling the initial stages of JEV infection.

[Alpha interferon, antiviral proteins and their value in clinical medicine]. / Interféron alpha, protéines antivirales et applications médicales.

Type I interferon system is an important part of host's innate defense mechanisms against viral infections. The type I interferons mediate in part their antiviral effect via induction of various proteins. Among them the most widely known are 2'-5' oligoadenylate synthetase (2'-5' OAS) and a protein kinase (PKR). MxA, an other antiviral protein, is specifically induced by the type I interferons. The MxA protein contains the dynamin signature, which is implicated in transport processes. The MxA protein appears to block the replication of certain viruses at poorly defined steps. There are substantial differences in the antiviral activity of MxA between virus types. Indeed, the replication of vesicular stomatitis virus and influenza virus is inhibited by MxA, but not the one of type I herpes simplex virus. Measurements of interferon alpha and MxA levels may be of high value in clinical practice. Interferon alpha can be detected by using a bioassay based on the interferon alpha ability to protect cultured cells from the cytopathic effect caused by a selected challenged virus, or by using immunological techniques. The current bioassays are the most sensitive methods but they are cumbersome and lengthy, even though simplifications have been proposed. Immunological techniques are easier, however they do not explore the biological activity of the circulating interferon. The presence of type I interferon in biological samples (serum, plasma, cerebro-spinal fluid, cultured cell supernatants) can be indirectly assessed by capability of interferon alpha to induce in vitro the synthesis of MxA in a dose dependent manner in cultured cells. Following to the lysis of the cells, the induced MxA can be quantitated and hence the type I-interferon concentration can be determinated in samples. The quantitation of MxA protein in peripheral blood lysates can be useful as a specific marker of acute viral infections. A minute amount of whole blood (15 mul) is sufficient which facilitates its use in pediatrics. The specifically type-I-interferons inducible MxA protein is also a potential useful marker in the management of interferon alpha-treatment. Moreover, the detection of interferon alpha and antiviral proteins constitute an indirect approach for investigating the hypothesis of the role of viruses in chronic diseases with suspected infectious aetiology.

Bizelesin, a bifunctional cyclopropylpyrroloindole alkylating agent, inhibits simian virus 40 replication in trans by induction of an inhibitor.

Bizelesin, a bifunctional DNA minor groove alkylating agent, inhibits both cellular and viral (SV40) DNA replication in whole cells. Bizelesin inhibition of SV40 DNA replication was analyzed in SV40-infected cells, using two-dimensional (2D) neutral agarose gel electrophoresis, and in a cell-free SV40 DNA replication assay. Within 1 h of bizelesin addition to infected cells, a similar rapid decrease in both the level of SV40 replication intermediates and replication activity was observed, indicating inhibition of initiation of SV40 DNA replication. However, prolonged bizelesin treatment (>/=2 h) was associated with a reduced extent of elongation of SV40 replicons, as well as the appearance on 2D gels of intense spots, suggestive of replication pause sites. Inhibition of elongation and induction of replication pause sites may result from the formation of bizelesin covalent bonds on replicating SV40 molecules. The level of in vitro replication of SV40 DNA also was reduced when extracts from bizelesin-treated HeLa cells were used. This effect was not dependent upon the formation of bizelesin covalent bonds with the template DNA. Mixing experiments, using extracts from control and bizelesin-treated cells, indicated that reduced DNA replication competence was due to the presence of a trans-acting DNA replication inhibitor, rather than to decreased levels or inactivation of essential replication factor(s).

Endogenous salivary inhibitors of human immunodeficiency virus.

The human immunodeficiency virus type 1 (HIV-1) is rarely transmitted through salivary secretions, due in part to the presence of endogenous inhibitors. Here, the protective characteristics of the intraoral environment are summarized and inhibitory factors that reduce HIV-1 infectivity in vitro described, focusing on secretory leucocyte protease inhibitor (SLPI), a 12-kDa mucosal protein that blocks HIV infection in several cell-culture systems. SLPI appears to interact with a cellular surface molecule to limit viral entry into target cells. To determine whether the inhibitor has a similar role in vivo, the contribution of salivary SLPI to anti-HIV-1 activity was assessed. Whole unstimulated filtered salivas from infected and uninfected donors contained similar concentrations of the inhibitor. Depletion from SLPI filtered saliva produced a corresponding loss of inhibitory activity. In general, filtered whole salivas obtained from 10 donors had antiviral activities that correlated positively with SLPI concentrations. However, some samples having SLPI well below the concentration required for inhibitory activity in vitro exhibited modest inhibition, suggesting the presence of other anti-HIV-1 components in oral fluids. Thus, SLPI is a major but not sole inhibitor of this virus in saliva.

Differential effects of intrauterine and subcutaneous administration of recombinant ovine interferon tau on the endometrium of cyclic ewes.

Interferon tau (IFNtau) is the antiluteolytic signal produced by the conceptus of ruminants. Intrauterine administration of recombinant ovine IFNtau suppresses expression of endometrial estrogen receptor (ER) and oxytocin receptor (OTR) in the luminal and superficial glandular epithelia to abrogate the production of luteolytic prostaglandin F(2alpha) (PGF(2alpha)) pulses. Subcutaneous (s.c.) injections of recombinant ovine (o) IFNtau appear to extend the interestrous interval by altering uterine PGF(2alpha) response to oxytocin. The present study tested the hypothesis that antiluteolytic effects of roIFNtau injected into the uterine lumen (paracrine) or s.c. (endocrine) are equivalent in suppressing expression of endometrial ER and OTR and inducing uterine expression of type I IFN-regulated Mx and ubiquitin cross-reactive proteins (UCRP). Sixteen cyclic ewes were fitted with uterine catheters on Day 5 (Day 0 = estrus), were assigned randomly to receive treatment with control proteins or roIFNtau (2 x 10(7) antiviral units/day) by either intrauterine or s.c. injections from Days 11 to 15, and were ovariohysterectomized on Day 16. Results indicated that expression of ER and OTR mRNAs in endometrial epithelium was suppressed by intrauterine but not by s.c. injections of roIFNtau. Intrauterine injections of roIFNtau increased expression of Mx and UCRP mRNA in the endometrium. Subcutaneous injections of roIFNtau increased endometrial Mx mRNA levels but not UCRP mRNA. Unexpectedly, intrauterine and s.c. injections of roIFNtau were equally effective in inducing expression of Mx and UCRP mRNA in the corpus luteum. Although s.c. injections of roIFNtau induced Mx mRNA in the endometrial epithelium, s.c. injections of roIFNtau did not abrogate activation of the uterine luteolytic mechanism by suppressing epithelial ER and OTR expression. Therefore, results of this study failed to support the assumption that endocrine roIFNtau mimics antiluteolytic effects of paracrine IFNtau to improve pregnancy rates in sheep.