Purification and Characterization of Antibodies in Single-Chain Format against the E6 Oncoprotein of Human Papillomavirus Type 16.

In Human Papillomaviruses- (HPV-) associated carcinogenesis, continuous expression of the E6 oncoprotein supports its value as a potential target for the development of diagnostics and therapeutics for HPV cancer. We previously reported that the I7 single-chain antibody fragment (scFv) specific for HPV16 E6, expressed as an intrabody by retroviral system, could inhibit significantly the growth of cervical cancer cells in vitro and was even able to reduce tumor development in experimental HPV-related cancer models. Nevertheless, for the development of therapeutic tools to be employed in humans, it is important to achieve maximum safety guarantee, which can be provided by the protein format. In the current study, two anti-16E6 scFvs derived from I7 were expressed in E. coli and purified in soluble form by affinity chromatography. Specificity, sensitivity and stability in physiologic environment of the purified scFvs were demonstrated by binding studies using recombinant 16E6 as an antigen. The scFvs functionality was confirmed by immunofluorescence in cervical cancer cells, where the scFvs were able to recognize the nuclear E6. Furthermore, an antiproliferative activity of the scFvI7nuc delivered in protein format to HPV16-positive cell lines was observed. Our results demonstrate that functional anti-16E6 scFvs can be produced in E. coli, suggesting that such purified antibodies could be used in the diagnosis and treatment of HPV-induced malignancies.

Inhibition of HIV-1 replication by cyclopentenone prostaglandins in acutely infected human cells. Evidence for a transcriptional block.

Cyclopentenone prostaglandins (PGs) inhibit virus replication in several DNA and RNA virus models, in vitro and in vivo. In the present report we demonstrate that the cyclopentenone prostaglandins PGA(1) and PGJ(2) at nontoxic concentrations can dramatically suppress HIV-1 replication during acute infection in CEM-SS cells. PGs did not affect HIV-1 adsorption, penetration, reverse transcriptase activity nor viral DNA accumulation in HIV-1 infected cells. A dramatic reduction in HIV-1 mRNA levels was detected up to 48-72 h after infection (p.i.) in PG-treated cells, and HIV-1 protein synthesis was greatly reduced by a single PG-treatment up to 96 h p.i. Repeated PGA(1)-treatments were effective in protecting CEM-SS cells by the cytopathic effect of the virus, and in dramatically reducing HIV-1 RNA levels up to 7 d after infection. The antiviral effect was not mediated by alterations in the expression of alpha-, beta-, or gamma-interferon,TNFalpha, TNFbeta, IL6, and IL10 in HIV-infected CEM-SS cells. The fact that prostaglandins are used clinically in the treatment of several diseases, suggests a potential use of cyclopentenone PGs in the treatment of HIV-infection.

Cystamine potently suppresses in vitro HIV replication in acutely and chronically infected human cells.

We have investigated the effects of cystamine on the replication of human immunodeficiency virus (HIV) in human lymphocytes and macrophages, the natural targets of HIV in vivo. Treatment of chronically infected macrophages with cystamine, at a concentration (500 microM) that did not show any cytotoxic or cytostatic effects, strongly decreased (> 80%) HIV-p24 antigen production and completely abolished the production of infectious viral particles. Cystamine does not affect viral transcription, translation or protein processing; indeed, all HIV proteins are present in a pattern similar to that of nontreated cells. Instead, cystamine interferes with the orderly assembly of HIV virions, as shown by electron microscopy analysis, that reveals only defective viral particles in treated cells. Moreover, suppression of HIV replication, due to the inhibition of proviral DNA formation was observed in acutely infected lymphocytes and macrophages pretreated with cystamine. These results show that cystamine potently suppresses HIV replication in human cells by contemporaneously blocking at least two independent steps of the viral life cycle, without affecting cell viability, suggesting that this compound may represent a new possibility towards the treatment of HIV-1 infection.

Modulation of prostaglandin A1-induced thermotolerance by quercetin in human leukemic cells: role of heat shock protein 70.

Prostaglandins of the A type (PGAs) function as signals for heat shock protein (hsp) synthesis in mammalian cells. In human K562 erythroleukemic cells, PGA1 induces the synthesis of a M(r) 70,000 hsp (hsp70) by cycloheximide-sensitive activation of heat shock transcription factor (HSF). Induction of hsp70 has been associated recently with the ability of PGA to protect K562 cells from thermal injury, establishing a thermotolerant state; however, the role of hsp70 in thermotolerance is still controversial. Because quercetin was shown to modulate hsp70 expression after heat shock in K562 cells, we have investigated the effect of this flavonoid on HSF activation, hsp70 synthesis, and thermotolerance in human K562 cells after induction with PGA1. Quercetin was found to inhibit hsp70 synthesis for a period of 3-6 h after PGA1 treatment. This transient block was exerted at the transcriptional level and was not due to the loss of HSF DNA-binding activity. After the initial delay, hsp70 synthesis reached the same rate as the PGA1-treated control, and it was actually prolonged in the presence of quercetin. In PGA1-treated cells, quercetin suppressed PGA1-induced thermotolerance completely if the heat shock was applied at a time (6 h) when hsp70 synthesis was inhibited, whereas it could not prevent the establishment of a thermotolerant state if the heat challenge was applied 24 h after treatment, when hsp70 synthesis was not affected. These results support strongly the hypothesis that hsp70 is involved in the establishment of thermotolerance in human cells.

Modulation of the growth of a human erythroleukemic cell line (K562) by prostaglandins: antiproliferative action of prostaglandin A.

Among several prostaglandins (PGs) tested, PGF1 alpha was found to slightly enhance, while PGAs and PGDs were found to drastically inhibit the growth of K562 cells in culture. This effect was dose dependent. While PGD2 was cytotoxic, PGA1 treatment could totally inhibit K562 cell proliferation, without affecting cell viability. PGA1 action was reversible; however, K562 cells totally lost their growth potential after prolonged exposure to PGAs. While it did not significantly affect DNA and RNA synthesis, PGA1 partially inhibited protein synthesis and glycosylation in these cells. In particular, the production of two polypeptides with molecular weights of 92,000 and 46,000, respectively, was decreased, while the synthesis of a protein with a molecular weight of 74,000 was induced. These results strongly support the concept that PGs are involved in the regulation of cell proliferation, and that PGs containing a reactive alpha, beta-unsaturated carbonyl group in the cyclopentane ring are potential antineoplastic agents.

Aspirin enhances thermotolerance in human erythroleukemic cells: an effect associated with the modulation of the heat shock response.

Heat shock protein (HSP) synthesis is induced by hyperthermia and other types of stress in mammalian cells in vitro and in vivo. In the present report we describe that in human erythroleukemic cells, aspirin (400 microM), when administered during or immediately after a hyperthermic treatment, causes an increase in the amount of HSP70 synthesized and prolongs HSP70 synthesis for a period of several hours. This effect is not due to increased HSP70 mRNA stability. In the presence of aspirin, the heat shock transcription factor is maintained in the activated DNA-binding state for a period twice as long as control, an effect which results in enhanced and prolonged HSP70 mRNA transcription. A different cyclooxygenase inhibitor, indomethacin (10(-7) M), also provokes similar effects. The modulation of the heat shock response by aspirin and indomethacin is associated with the ability of these drugs to potentiate the effect of hyperthermia and prolong thermotolerance for a period of 48 h. These results indicate that the use of aspirin and indomethacin should be carefully monitored in cancer patients undergoing hyperthermic treatment. On the other hand, the ability of aspirin to enhance HSP70 synthesis suggests that nonsteroidal anti-inflammatory drugs could potentiate the cytoprotective role of HSPs in pathological states, including fever, inflammation, and ischemia.

Herpes simplex virus 2 causes apoptotic infection in monocytoid cells.

Increasing evidence indicates that apoptosis can be associated with several viral infections. Here we demonstrate, that infection of monocytoid cells by Herpes simplex virus 2 (HSV-2) resulted, in time- and dose-dependent induction of apoptosis as an exclusive cytopathic effect. The phenomenon was confirmed using four different techniques. Conversely, apoptosis was not observed in the Vero cell line. Virus yield in monocytoid cells was delayed and reduced, although well detectable, in comparison with that observed in Vero cells. Nevertheless, released virions exhibited full infecting capability. Apoptosis induced by HSV-2 was not inhibited by cycloheximide and only partially by an UV-treatment which completely abrogated infectivity. Virus-induced apoptosis was partly inhibited by indomethacin and was associated with a down-regulation of Bcl-2. A similar, but less pronounced, apoptosis-inducing effect in monocytoid cells was also observed with HSV-1 infection. Depending on the target cells, therefore, HSV could complete a cycle of infection which is characterized by apoptosis of infected cells.

Antiproliferative activity of cyclopentenone prostaglandins in early HTLV-1 infection is independent of IL-2 and is associated with HSP70 induction.

Cyclopentenone prostaglandins PGA1 and PGJ2 can inhibit the growth of HTLV-1 infected cord blood-derived human mononuclear cells (CBMC), both after acute infection and in chronically infected, immortalized cells. When CBMC were exposed to HTLV-1 infection by coculturing with lethally irradiated, virus-donor allogeneic MT-2 cells, they underwent a proliferative response, that peaked within the first week and then declined. PG treatment did not inhibit the initial proliferation (day 4) of cocultured CBMC, while multiple treatments with PGA1 and more efficiently with PGJ2, suppressed the late cell proliferation (from day 8 onward). The pharmacological effects of PGA1 and PGJ2 were reversible and therefore multiple treatments were required to maintain their antiproliferative activity. Increasing concentrations (20, 40, 80 IU/ml) of recombinant IL-2 did not affect the virus-associated proliferative response of CBMC, and exogenous IL-2 did not revert the antiproliferative effect of both PGs. Arrest of proliferation in cocultured CBMC occurred concomitantly with expression of high levels of HSP70 in the cells. In fact, though HSP70 expression was induced early (day 5) after exposure to HTLV-1, its expression was further increased after multiple PG treatments and high levels were found when the antiproliferative effect of PGs became manifest. Since HSP70 protein family is involved in the control of cell cycle as well as in antigen processing and presentation during the immune response against tumor cells and pathogens, the persistent expression of this protein in PG-treated cocultures suggested that, beside inhibiting the growth of virus-infected cells, HSP70 expression might play a role in modulating the immune function of CBMC. However, unlike in most virus infection models, in which cyclopentenone PGs exert clear antiviral effects by inhibiting the synthesis and maturation of virus proteins, no antiviral activity was found in this model of infection. This strongly suggests that the main effect of these PGs against HTLV-1 infected cells consists in inhibiting proliferation in vitro without affecting viral expression.

Wheat germ agglutinin-binding protein changes in highly malignant Friend leukemia cells metastasizing to the liver.

We have used binding of radioactive lectins (i.e. Concanavalin A (ConA), Wheat Germ Agglutinin (WGA) and Ricinus communis agglutinin I (RCAI)) to membrane glycoproteins separated in SDS gel electrophoresis, to detect specific carbohydrate changes in plasma membrane proteins of in vivo passaged Friend erythroleukemia cells (FLC). These cells are highly metastatic to the liver, whereas the original in vitro passaged tumor cells do not metastasize. Marked qualitative differences in the high molecular weight region of the gels (100-200 kD) were observed between the WGA binding glycoproteins of metastatic in vivo passaged FLC and nonmetastatic in vitro passaged FLC. Furthermore, the binding of WGA to plasma membrane proteins of in vivo passaged FLC was much greater than the binding of WGA to plasma membrane proteins of in vitro passaged FLC. Lectin binding experiments after sialic acid removal by in situ mild acid hydrolysis of FLC glycoproteins indicated that an increased sialylation of the 120 and 145 kD glycoproteins was responsible for the increased WGA reactivity of in vivo passaged FLC plasma membranes. Besides the increased sialylation, other changes in glycosylation of the 100-200 kD glycoproteins of in vivo passaged FLC were observed: (1) qualitative differences between the WGA binding patterns of the two cell types were restored after treatment of the gels with mild acid and subsequent Smith degradation; (2) after chemical removal of sialic acid residues from the gels, qualitative differences in the RCA binding patterns to the glycoproteins of the two cell types were apparent.

Cell mediated cytotoxicity and cytokine production in peripheral blood mononuclear cells of glioma patients.

A mannoprotein preparation (MP) from Candida albicans induced MHC-unrestricted cytotoxicity in peripheral blood mononuclear cells (PBMC) from healthy subjects, but not in those from glioma-bearing subjects. The two groups of subjects did not significantly differ in the number of cells bearing typical natural killer (NK) markers (both in resting and MP stimulated PBMC) and NK activity. However, interferon gamma (IFN-gamma) production was in tumour patients minimal or significantly reduced, as compared to healthy subjects, following PBMC stimulation by MP or phytohaemoagglutinin, respectively. In addition, minimal, if any, stimulation of interleukin-2 (IL-2) production was achieved in MP stimulated PBMC from glioma patients. Considering the pivotal role of the above cytokines in immune responses, particularly in those concerning generation of antitumour effectors, our results consistently suggest that defective cytokine production is one possible mechanism of immunological impairment in glioma patients. They also provide indirect support for a possible clinical use of IFN-gamma as an immunopotentiating agent in gliomatous subjects.

Sendai virus replication in Friend erythroleukemia cells. I. Acutely and persistently infected cells become resistant to virus-induced lysis.

Friend leukemia cells (FLC) are susceptible to infection by Sendai virus, a member of the paramyxovirus group. FLC constitute a most suitable model to study virus-host cell interactions, because they grow in suspension (thus avoiding the use of trypsin), and provide an easy way of deriving single-cell clones. When FLC are infected with Sendai virus at high m.o.i., a direct, extensive lysis of the cells ensues, whereas lower doses of virus result in a cytocidal infection whose lethality depends mainly on the virus used, standard or defective interfering egg-grown Sendai virus (EGSV), and on the multiplicity of infection (m.o.i). At later times after infection, FLC become resistant to the Sendai induced lysis (SIL). The SIL resistance can be maintained in single-cell clones that had survived the first infection. The maintenance of the resistant phenotype of the clones requires the serial subcultivation of the cells in the presence of activated EGSV. The mechanisms that presumably regulate the appearance of SIL resistance in Sendai infected FLC are discussed.

HSP70 production and inhibition of cell proliferation in Molt-4 T-cells after cell-to-cell transmission of HTLV-I: effect of PGA1.

Infection with HTLV-I is associated with leukemic transformation of mature CD4+ T lymphocytes. PGA1, a powerful inhibitor of tumour cell proliferation, can prevent the clonal expansion of HTLV-I-infected cells following acute infection of cord blood-derived mononuclear cells. Since the antiproliferative effect of PGA1 on HTLV-I transformed, chronically infected MT-2 cell line was associated with induction of HSP70, we have investigated the effect of PGA1 on cell cycle progression and HSP70 production in a leukemic T-cell line (Molt-4) shortly after exposure to HTLV-I in a cell-to-cell transmission model. Rate of cell proliferation and HSP70 expression were studied within one duplication cycle of Molt-4 cells after exposure to HTLV-I. Growth of both control and virus-exposed cultures was inhibited by treatment with PGA1 (4 micrograms/ml) and cell cycling was arrested preferentially at the G1/S interphase. Synthesis of HSP70 was induced within 3 h by PGA1 in control and virus-exposed Molt-4 cells and became undetectable from overnight onward, though the protein accumulated in the cells. The arrest of growth was observed from overnight up to 48 h so that treated cells almost missed one cycle. Interestingly, HSP70 transcript and protein persisted at remarkably high levels in Molt-4 cells exposed to HTLV-I in the absence of PGA1, showing that HSP70 expression can be directly activated during primary infection with this human retrovirus. Moreover, in these cocultures, treatment with PGA1 or heat shock was not able to increase further the elevated level of HSP70 found in untreated cocultures, suggesting that during the early period of the virus-transmission phase, HTLV-I could interfere with HSP70 induction by other inducers.

Inhibition of Sendai virus replication by delta 12-prostaglandin J2: induction of heat shock protein synthesis and alteration of protein glycosylation.

delta 12-Prostaglandin J2 (delta 12-PGJ2), a natural dehydration product of prostaglandin D2 present in human body fluids, was shown to suppress Sendai virus replication in monkey kidney cells, at doses non-toxic to uninfected cells. Dramatic inhibition of virus production could be obtained at doses of delta 12-PGJ2 which did not inhibit cellular or viral protein synthesis, suggesting an effect on virus assembly and/or maturation. At the active concentration, delta 12-PGJ2 caused a decrease in glucosamine incorporation into the virus glycoproteins HN and F, and in at least one host cell polypeptide, while it did not affect most cellular glycoproteins and it induced the glycosylation of a 47-kDa cellular polypeptide. These effects were accompanied by the induction of heat shock protein synthesis, which was found to differ in its specificity and kinetics from induction by prostaglandin A1.

Microtubule assembly in Friend erythroleukemia cells spread on fibronectin- and lectin-coated substrates.

A comparison was carried out between parental Friend Erythroleukemia cells (FLC, 745 A clone) and highly fibronectin (FN)-sensitive clones of FLC for their ability to adhere, spread and organize microtubular (MT) apparatus, when seeded on FN- or lectin-coated plastic substrates. While FN was able to induce the spreading only in the FN-sensitive FLC clones (further referred to as FF clones) and not in the parental 745 A cells, the lectins Concanavalin A (ConA) and Leukoagglutinin (LeuA) promoted the spreading of both 745 A and FF cells, with no differences between the two cell lines. Wheat germ agglutinin (WGA), instead, is almost ineffective in triggering cell spreading in both cell clones. The spreading of FLC, either 745 A or FF, on any of the ligands tested, is always accompanied by a massive reorganization of the MT apparatus of the cell. Possible mechanisms involved in the selective spreading effect, exerted by FN, are discussed.

A nonneutralizing human IgM monoclonal antibody inhibiting hemagglutination of H3N2 influenza A strains.

A mouse-human hybridoma has been produced by fusing human splenocytes from a Cooley's anemia patient with the murine myeloma P3-NS1/1-Ag 4-1. The hybridoma is stable after 18 months and secretes human IgM. The antibody reacts with some H3N2 influenza A strains and detects an epitope that is part of the hemagglutinin antigen, but does not affect virus infectivity.