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Proteolytic enzymes (also termed peptidases, proteases and proteinases) are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics. The aim of this paper is to review the molecular biological aspects of proteolytic enzymes and summarize their applications in the life sciences.

The capsid protein of the human immunodeficiency virus type 1 has been found to be a substrate of the retroviral protease in vitro, and its processing was predicted to be strongly dependent on a pH‐induced conformational change. Several protease cleavage sites have been identified within the capsid protein, but the importance of its cleavage by the viral protease at the early phase of infection is controversial. To confirm the relevance of this process, we aimed to design, produce, and characterize mutant capsid proteins, in which the protein susceptibility toward HIV‐1 protease is altered without affecting other steps of the viral life cycle. Our results indicate that while the introduced mutations changed the cleavage rate at the mutated sites of the capsid protein by HIV‐1 protease, some of them caused only negligible or moderate structural changes (A78V, L189F, and L189I). However, the effects of other mutations (W23A, A77P, and L189P) were dramatic, as assessed by secondary structure determination or cyclophilin A‐binding assay. Based on our observations, the L189F mutant capsid remains structurally and functionally unchanged and may therefore be the best candidate for use in studies aimed at better understanding the role of the protease in the early postentry events of viral infection or retrovirus‐mediated gene transduction.

Replication of human cytomegalovirus (HCMV) was investigated in various T-cell lines expressing the tax gene product of human T-cell leukemia-lymphoma virus type I (HTLV-I). Differential patterns of HCMV replication were found in HTLV-I-carrying cell lines. HCMV gene expression was restricted to the immediate-early genes in MT-2 and MT-4 cells, whereas full replication cycle of the virus was observed in C8166-45 cells. Productive HCMV infection induced a cytopathic effect resulting in the lysis of infected cells. The results of electrophoretic mobility shift assay (EMSA) showed high levels of NF-kappaB-, CREB/ATF-1-, and SRF-specific DNA binding activity in all Tax-positive cell lines. In contrast, SP1 activity could be detected only in C8166-45 cells. Using an inducible system (Jurkat cell line JPX-9), a dramatic increase in NF-kappaB, CREB/ATF-1, SRF, and SP1 binding activity, as well as productive HCMV infection, were observed upon Tax expression. Overexpression of SP1 in MT-2 and MT-4 cells converted HCMV infection from an abortive to a productive one. These data suggest that the stimulatory effect of Tax protein on HCMV in T cells is accomplished through at least five host-related transcription factor pathways. The results of this study provide possible mechanisms whereby HCMV infections might imply suppression of adult T-cell leukemia.

Human herpesvirus 6 (HHV-6) and human immunodeficiency virus type 1 (HIV-1) may interact during transplacental transmission of HIV-1. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. Patterns of replication of HHV-6 variant A (HHV-6A) and HIV-1 were analyzed in singly and dually infected human term syncytiotrophoblast cells cultured in vitro. For this purpose, the GS strain of HHV-6A and the Ba-L and IIIB strains of HIV-1 were used. HHV-6A replication was restricted at the level of early gene products in singly infected syncytiotrophoblasts, whereas no viral protein expression was found in cells infected with HIV-1 alone. Coinfection of syncytiotrophoblast cells with HHV-6A and HIV-1 resulted in production of infectious HIV-1. In contrast, no enhancement of HHV-6A expression was observed in cell cultures infected with both viruses. Uninfected syncytiotrophoblast cells were found to express CXCR4 and CCR3 but not CD4 or CCR5 receptors. Infection of syncytiotrophoblasts with HHV-6A did not induce CD4 expression and had no influence on chemokine receptor expression. Activation of HIV-1 from latency in coinfected cells was mediated by the immediate-early (IE)-A and IE-B gene products of HHV-6A. Open reading frames U86 and U89 of the IE-A region were able to activate HIV-1 replication in a synergistic manner. The data suggest that in vivo double infection of syncytiotrophoblast cells with HHV-6A and HIV-1 could contribute to the transplacental transmission of HIV-1 but not HHV-6A.

Sensitive detection methods, such as DNA PCR and RNA PCR suggest that vertical transmission of human immunodeficiency virus (HIV) occurs at three major time periods; in utero, around the time of birth, and postpartum as a result of breastfeeding (Fig. 1). Detection of proviral DNA in infant's blood at birth suggests that transmission occurred prior to delivery. A working definition for time of infection is that HIV detection by DNA PCR in the first 48 h of life indicates in utero transmission, while peripartum transmission is considered if DNA PCR is negative the first 48 h, but then it is positive 7 or more days later [1]. Generally, in the breastfeeding population, breast milk transmission is thought to occur if virus is not detected by PCR at 3-5 months of life but is detected thereafter within the breastfeeding period [2]. Using these definitions and guidelines, studies has suggested that in developed countries the majority, or two thirds of vertical transmission occur peripartum, and one-third in utero [3-6]. The low rate of breastfeeding transmission is due to the practice of advising known HIV-positive mothers not to feed breast milk. However, since the implementation of antiretroviral treatment in prophylaxis of HIV-positive mothers, some studies have suggested that in utero infection accounts for a larger percentage of vertical transmissions [7]. In developing countries, although the majority of infections occurs also peripartum, a significant percentage, 10-17%, is thought to be due to breastfeeding [2, 8, 9].

Intrauterine infection of the fetus is clearly an important mode of vertical transmission of human immunodeficiency virus type 1 (HIV-1). The syncytiotrophoblast layer of the human placenta must be traversed by HIV-1 in order to reach underlying cells and fetal capillaries. Although HIV-1 has been detected in the syncytiotrophoblast layer in situ, there is conflicting evidence regarding infection of syncytiotrophoblast cells with cell-free virus. The phenotypic mixing between HIV-1 and vesicular stomatitis virus (VSV) has been exploited to assay the susceptibility of human term syncytiotrophoblast cells to penetration by various strains of HIV-1. VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) pseudotypes were found to enter syncytiotrophoblast cells. In contrast, VSV pseudotyped with envelope glycoproteins of RF, MN, or Ada-M strains of HIV-1 did not infect syncytiotrophoblasts. Plating efficiency of VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) was 10-fold lower on syncytiotrophoblasts than on T-cells and macrophages, respectively. Incubation of VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) viruses with appropriate HIV-1 neutralizing sera before infection strongly inhibited entry of pseudotyped VSV into syncytiotrophoblast cells. These findings demonstrated that infection of syncytiotrophoblasts with VSV(HIV-1) pseudotypes was mediated by Env from IIIB and Ba-L strains of HIV-1. Monoclonal antibodies (MAb) to CD4, CXCR4, CCR5, and CCR3 were tested for their ability to block VSV(HIV-1) infection of syncytiotrophoblast cells. Neither the anti-CD4 nor the anti-CXCR4, anti-CCR5, and anti-CCR3 MAb had any inhibitory effect on infection of syncytiotrophoblast cells with VSV(HIV-1) pseudotypes. Results from this study suggest that cell-free HIV-1 can enter syncytiotrophoblasts and the susceptibility of these cells to penetration by the virus is strain dependent. Pseudotype infection merely demonstrates that the first steps in HIV-1 replication are possible in syncytiotrophoblast cells.

Antibodies to solid phase C1q (C1qAb) were determined in 295 serum samples from 132 HIV-infected subjects and in sera from 140 HIV-seronegative healthy individuals as control. An ELISA method applied for the determination of C1qAb in other diseases was used. In part of these sera, other autoantibodies (antibodies reacting with 60-kDa human heat shock protein (hsp60) or mycobacterial hsp65; IgA and IgG class antibodies against the Fab and F(ab′)2 moieties of IgG) as well as complement-mediated antibody-dependent enhancement/neutralization (C′-ADE) were also determined. Increased amount of C1qAb was found in HIV-infected subjects as compared with HIV-seronegative controls (P= 0.0138). In 17 of 132 (13.0%) seropositive individuals but only in 7/140 (5.0%) samples from the controls, the amount of C1qAb exceeded the upper limit (95th percentile) of the normal values (P= 0.031). The amount of C1qAb significantly decreased during a follow-up period of 65 months. C1qAb levels were found to strongly correlate to hsp60/65 autoantibodies but did not correlate or only weakly correlated to the amount of anti-Fab or anti-F(ab′)2autoantibodies measured in the same serum samples. Anti-C1q antibodies recognized the solid phase hsp60/65. Three predicted epitope regions ofM. paratuberculosishsp65 were able to bind efficiently C1q antibodies. An inverse correlation was found between C1qAb and C′-ADE, neutralization was more frequent in the sera with detectable C1qAb, whereas sera without C1qAb more likely enhanced HIV infectionin vitro.

Summary Although both human immunodeficiency virus type 1 (HIV-1) and human T cell leukemia-lymphoma virus type I (HTLV-I) can enter syncytiotrophoblast (ST) cells, the trophoblast is assumed to restrict the rate of retrovirus transmission. We studied the interactions of human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) with HIV-1 and HTLV-I in human term ST cells. HCMV replication was restricted at the level of immediate-early (IE) and early (E) gene products in singly infected cells. Previous or simultaneous infection of the cells with HCMV resulted in marked upregulation of HIV-1 replication, whereas prior HIV-1 infection of the cells converted HCMV replication from nonpermissive to permissive in nature. In contrast, coinfection of ST cells with HCMV and HTLV-I resulted in simultaneous replication of both viruses. Enhancing activities between retroviruses and HCMV were mediated primarily by the Tat, Tax and IE proteins. The ST cells could be productively infected with EBV, and dual infection of the cells with EBV and HTLV-I resulted in full replication cycle of otherwise latent HTLV-I. Permissive replication cycle of HTLV-I was induced by the EBV IE gene product Zta. Our data suggest that HCMV and EBV may play a critical role in the transplacental transmission of human retroviruses.

Summary The human cytotrophoblast forms the interface between mother and fetus. It undergoes differentiation through two multi-step pathways, resulting in the formation of invasive (extravillous) and non-invasive (villous) trophoblast populations. These trophoblast populations produce interferons (IFNs) of the alpha, beta, omega and gamma-like types when infected with viral and non-viral inducers. The type and level of IFN production is dependent on the trophoblast population, type of inducer and the stage of differentiation. First trimester extravillous trophoblast cultures produce greater than five-fold more IFN than third trimester villous trophoblast on a per cell basis, whereas syncytiotrophoblast produces twice as much IFN as term mononuclear villous trophoblast. The trophoblast IFNs have antiviral, antiproliferative and immunosuppressive properties, and furthermore activate natural killer cell activity in vitro . The trophoblast IFNs inhibited the expression of proto-oncogenes (EGF-R, c-erbB2, and c-fms/CSF-1R) that are reported to play a role in trophoblast proliferation, differentiation and invasion. These data may suggest the possible functions of human trophoblast IFNs during embyronic development with respect to regulation of growth and differentiation, protection of the fetus against viral infection and maternal immunity.

The human cytotrophoblast is the first fetal cell type to arise during embryogenesis and differentiate along two pathways to the invasive (extravillous) and non-invasive (villous) populations. The non-invasive villous trophoblast differentiate morphologically and biochemically to form terminally differentiated multinucleated syncytial trophoblast. First trimester invasive and non-invasive trophoblast were isolated from human placentae (5-12 weeks) and were cultured in vitro. The villous trophoblast cells differentiated in vitro to form aggregated syncytial cells which was associated with increased expression of epidermal growth factor receptor (EGF-R). The invasive trophoblast cells expressed colony-stimulating factor receptor (c-fms/CSF-1R) and c-erbB2 proteins but low levels of EGF-R. We studied the effects of human trophoblast-induced interferon (IFN)-alpha/beta on the expression of c-fms/CSF-1R, EGF-R and c-erbB2 whose ligands are reported to be involved in the regulation of growth and differentiation of normal invasive and non-invasive trophoblast cells. Human trophoblast-induced IFN-alpha/beta (100 IU/ml) reduced the expression of EGF-R in both invasive and non-invasive trophoblast cells as determined by quantitative enzyme-linked immunosorbant assay ('ELISA') and western immunoblot methods. The same amount of IFN activity reduced the expression of c-fms/CSF-1R and c-erbB2 proto-oncogene products in invasive trophoblast cells. These results may suggest a possible role of trophoblast-induced IFNs in the regulation of normal trophoblast growth, differentiation and function.

Human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1) may interact in the pathogenesis of AIDS. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. We analyzed the patterns of replication of HIV-1 and HCMV in singly an dually infected human term syncytiotrophoblast cells cultured in vitro. Syncytiotrophoblast cells exhibited restricted permissiveness for HIV-1, while HCMV replication was restricted at the level of immediate-early and early gene products in the singly infected cells. We found that the syncytiotrophoblasts as an overlapping cell population could be coinfected with HIV-1 and HCMV. HIV-1 replication was markedly upregulated by previous or simultaneous infection of the cells with HCMV, whereas prior HIV-1 infection of the cells converted HCMV infection from a nonpermissive to a permissive one. No simultaneous enhancement of HCMV and HIV-1 expression was observed in the dually infected cell cultures. Major immediate-early proteins of HCMV were necessary for enhancement of HIV-1 replication, and interleukin-6 production induced by HCMV and further increased by replicating HIV-1 synergized with these proteins to produce this effect. Permissive replication cycle of HCMV was induced by the HIV-1 tat gene product. We were unable to detect HIV-1 (HCMV) or HCMV (HIV-1) pseudotypes in supernatant fluids from dually infected cell cultures. Our results suggest that interactions between HIV-1 and HCMV in coinfected syncytiotrophoblast cells may contribute to the transplacental transmission of both viruses.

Diabetic retinopathy is the leading cause of new cases of legal blindness among adults in the developed countries. Approximately 40% of all people with diabetes have diabetic retinopathy and 5% of these have sight-threatening form. As the advanced stage, where there is a high risk for vision loss, can develop without any serious symptoms, sometimes it is hard to detect it. A non invasive method to detect biomarkers characteristic for diabetic retinopathy from the tear fluid was developed. Tear samples from diabetic patients with no retinopathy, non proliferative and proliferative stages of diabetic retinopathy were analyzed and the protein content of each sample was compared to the protein content of tear pool from healthy volunteers. The samples were labeled with iTRAQ fourplex labels and were analyzed with nanoHPLC coupled ESI-MS/MS mass spectrometry. The lipocalin 1, lactotransferrin, lacritin, lysozyme C, lipophilin A and immunoglobulin lambda chain were identified as possible biomarker candidates with significantly higher relative levels in the tear of patients with diabetic retinopathy.

Human placental trophoblast cells produce predominantly interferon-beta-type (IFN-beta) when stimulated with viral inducers. The aim of the present study was to determine the in vitro antiproliferative effect of the trophoblast interferon-beta (tro-IFN-beta) on mitogen-stimulated and resting lymphocytes. The antiproliferative effect of the tro-IFN-beta was compared to human recombinant IFN-beta. All activities of tro-IFN-beta and human recombinant IFN-beta ranging between 10-1000 IU/ml showed suppression of proliferative responses on mitogen-stimulated and resting lymphocytes compared to cultures without IFN treatment. The inhibitory level of both tro-IFN-beta and recombinant IFN-beta was significantly higher on the stimulated than on the resting lymphocytes. Although there was a variation in the inhibition of lymphocyte proliferation by both IFNs with respect to time, there was no statistically significant difference in the antiproliferative effect of the IFNs on both resting and mitogen-stimulated lymphocytes. Since IFNs are produced locally by the placenta during pregnancy, our data suggest that in addition to the antiviral activity, the human tro-IFN-beta may participate in the local control of the maternal immune response during pregnancy at the fetomaternal interface.

Summary Stimulation of human first and third trimester trophoblast and syncytotrophoblast cultures with viruses (Sendai and Newcastle Disease virus) led to the production of a mixture of IFN-α subtypes and-β. The magnitude and compositions of the IFNs produced were dependent on the gestational age of the trophoblast, the type of inducer and the stage of differentiation of the trophoblast. The data obtained indicated that the first trimester trophoblast cultures produce 5-to 6-fold more IFNs than the third trimester trophoblast on per cell basis whereas syncytitrophoblast produced 2-fold more IFNs than the mononuclear trophoblast at term when challenged with viruses. The viruses induced different levels and compositions of IFN-α and -β in both first and third trimester trophoblast and syncytiotrophoblast cultures. Tandem high-performance affinity chromatography (HPAC) of the virusinduced trophoblast interferon (tro-IFN) preparations resulted in the isolation of IFN-α subtypes with molecular masses of 16 and 22 kDa and β with molecular mass 24 kDa on SDS-PAGE. The antiviral activity of the tro-IFNs were stable at pH 2.0 and a fraction of the tro-IFN-α and -β were demonstrated to be glycoproteins. The tro-IFNs showed different antiviral activities when assayed on human and heterologous bovine cell species. Tro-IFN-α subtypes protected both human and bovine (MDBK) cells from virus infection whereas tro-IFN-β showed a high degree of species specificity being only active in protecting the human cell types tested. The tro-IFN-α and -β inhibited the secretion of prostaglandin E 2 (PGE 2 ) in trophoblast cultures in vitro .

The study was undertaken to determine if HSV infection of trophoblast cells would alter the general resistant state of trophoblast cells to MHC non-restricted cell-mediated cytotoxicity. The NK cell activity against HSV-infected human term placental trophoblast cells was investigated. In a 12-hour 51Cr release assay, PBMC, or immunosorted peripheral blood CD56+ NK cells from healthy donors were found not to lyse HSV-1-infected trophoblast cells, while HSV-1-infected placental fibroblast cells were preferentially lysed when using the same experimental set-up. Furthermore, using cold target competition assay, no inhibition of NK cell activity against the susceptible target cell K562 was noted when using HSV-infected or uninfected trophoblast cells. Inversely, both HSV-infected and uninfected fibroblast cells demonstrated a competitive inhibition of NK cell lysis of K562 cells. The observed differences between the susceptibility of HSV-infected placental trophoblast and fibroblast cells to NK lysis could not be explained by a difference in the surface expression of HSV-antigens or transferrin receptors. Additionally, no significant differences were observed for de novo production of IFN by effector cells during the cocultivation with HSV-infected trophoblast and fibroblast cells that would explain the observed discrepancies in NK susceptibility. Consequently, these in vitro results suggest that the observed resistance of trophoblast cells to maternal cell-mediated lysis is not modified by HSV-1 infection and that the peripheral blood NK cells are not involved in the prevention of a possible vertical HSV spread through infected trophoblast cells.

Forty patients with chronic granulocytic leukemia (CGL) were tested for antibodies and lymphocytes reacting with gibbon ape leukemia virus (GaLV) and baboon endogenous virus (BaEV) antigens as well as for plasma interferon levels. Antibodies reacting with envelope antigens of GaLV and BaEV were found frequently and in high titers in patients with the quiescent phase of CGL but rarely and in low titers in the accelerated and blastic phase of the disease. Results of radioimmunoprecipitation studies were in concordance with those obtained in virus neutralization experiments. Cellular and humoral cytotoxic activity of blood plasma and lymphocyte samples against autologous tumor cells showed a similar phase-specific distribution. Most of these activities could be blocked by GaLV and BaEV gp70 antigens. Elevated plasma interferon (IFN)-alpha levels were found in the quiescent and accelerated phase of CGL, whereas no significant differences could be detected between IFN levels of patients with the blastic crisis of CGL and those of the control persons. Follow up studies of four patients confirmed this stage-specific distribution of antiretroviral immune and interferon response.

Reclustering and indirect immunofluorescence assays on MT-4 cells [carrying both CD4 and complement receptor type 2 (CR2)] were used to measure neutralizing and enhancing antibodies in sera obtained from HIV-1-infected individuals. Heat-inactivated sera were tested before and after mixing 1:1 with fresh seronegative human serum. Using heated samples, neutralizing antibodies were found in 20 out of 20 and 11 out of 19 serum samples of asymptomatic and symptomatic [AIDS, AIDS-related complex (ARC)] HIV-seropositive patients, respectively. In complement-restored samples, neutralizing activity was found in eight sera of asymptomatic patients and in none of the sera of AIDS and ARC patients; enhancing activity could be detected in four and 12 sera, respectively. A significant positive correlation was observed between the titres of neutralizing antibodies measured in the complement-restored samples and the absolute number of CD4+ lymphocytes. These findings indicate that the appearance of complement-dependent enhancing antibodies coincident with the loss of neutralizing antibodies may indicate a poor prognosis in HIV infection.

We recently described a beta interferon response of primary cultures of term human trophoblast exposed to poly(I:C). The response pattern has now been studied further with priming and superinduction both of normal placental cell types and JAR, JEG-3 and BeWo choriocarcinoma cells. Pre-treating placental trophoblast cells, fibroblasts and macrophages with human interferon generally led to increased yields of interferon after poly(I:C) induction, whereas choriocarcinoma cells did not respond to priming. All the cells showed the superinduction phenomenon although to varying degrees. The combination of priming and superinduction conditions led to the production of very high yields of interferon in placental fibroblast cultures. The combined procedure also produced more interferon in macrophage cultures than priming or superinduction alone. Combined superinduction and priming of normal trophoblast did not produce higher yields than those obtainable by superinduction alone. These data might help to provide additional insight into the cellular control mechanisms of sensitivity of normal and malignantly transformed cells to interferon. Furthermore, the large quantity of trophoblast interferon produced by superinduction could be used for studies of its anti-viral and immunomodulatory effects.

Enhancement of in vitro infection by immune antibodies was described for several viral diseases, first for the dengue and respiratory syncytial virus infections. Immune enhancement of viral infection can be connected with vaccine failures since vaccinated individuals who develop enhancing antibodies may have an exaggerated disease upon exposure to the virus. A part of the antibody-dependent enhancement is mediated by complement activation; this enhancement is mediated by antibodies against the immunodominant part of the transmembrane protein (gp41) of HIV. Binding of the enhancing antibodies results in deposition of complement proteins (C1q) and activation products of C4 and C3 on virus particles; interaction of these proteins with the type 2 complement receptor (CR2) and the C1q receptor facilitates attachment of the HIV virions to the target cells and leads to increased virus production in vitro and probable in vivo as well. Complement mediated antibody dependent enhancement (C-ADE) was demonstrated to be clinically relevant in HIV infection by the authors of the present chapter. C-ADE was found to develop shortly after HIV-infection concomitantly with seroconversion, and strongly correlate to viral load in the course of HIV disease. C-ADE can be detected more frequently in advanced stages of HIV infection and their appearance may herald progression of disease. Combination treatment by antiviral drugs (HAART) leads to the disappearance of C-ADE; sera of the HAART-treated patients may neutralize HIV even in the presence of complement, which can be infrequently detected in untreated patients. These findings indicate that study of C-ADE may have of crucial importance at development of HIV vaccines.

Authors studied the effect of highly active antiretroviral therapy (HAART) on balance of the antibodies that enhance or neutralize growth of HIV-1(IIIB) strain in MT-4 cells in the presence or absence of human complement. Sequential serum samples were collected from 28 patients in advanced stage of HIV disease before and during HAART. The balance of the enhancing and neutralizing antibodies was expressed by an index value (E/N I). Samples with an E/N I of0.5 (twofold decrease in virus production) were considered as neutralizing, whereas samples with an E/N I2.0 (twofold increase in virus production) were considered as enhancing. At the beginning of HAART serum samples from eight patients enhanced, and samples from only two patients neutralized the virus in the presence of complement, median (25th-75th percentile) value of E/N I was 1.32 (0.79-2.29). E/N I significantly (P0.0001) dropped to 0.37 (0.19-0.57) during the follow-up period of 18.5 (10.5-23.5) months under HAART. Similar changes were detected when serum samples were tested with no complement added. The E/N I values were also markedly decreased when cultures inoculated with mixtures of HIV and purified IgG prepared from serum pools taken before and during HAART, respectively, were compared. In the last samples of 20/28 patients, neutralization was measured even in the presence of complement while enhancement was found with none of these samples. These findings suggest that HAART results in disappearance of enhancing antibodies and switches the E/N I toward neutralization.

The frequency and mechanism of p16(INK4A) and p14(ARF) gene alterations were studied in cell samples from 30 patients with Philadelphia (Ph) chromosome-positive chronic myeloid leukaemia (CML), both at diagnosis and at the onset of the accelerated phase (AP) of the disease. No alterations in the p16(INK4A) or p14(ARF) genes were found in any of the chronic phase (CP) samples. DNA sequencing analyses detected p16(INK4A) or p14(ARF) mutations in 17 AP samples. All mutations were heterozygous without loss of the other allele. Aberrant methylation of the p16(INK4A) or p14(ARF) promoters was found in 14 of 30 AP samples. The most common situation was the simultaneous methylation of both promoters. Our data indicate that p16(INK4A) and p14(ARF) are primary targets for inactivation by promoter methylation in the acceleration of CML. Transcriptional silencing of the p16(INK4A) and p14(ARF) genes may be important in the conversion of CML from the CP to the AP.

The human immunodeficiency virus (HIV) protease is a homodimeric aspartyl protease that is crucial for the viral life-cycle, cleaving proviral polyproteins, hence creating mature protein components that are required for the formation of an infectious virus. With diagnostic measures and clinically used protease inhibitors focusing on HIV-1, due to its higher virulence and prevalence, studies of the efficacy of those inhibitors on HIV-2 protease remain widely lacking. Utilizing a wild-type HIV-2 vector backbone and cloning techniques we have developed a cassette system where the efficacy of clinically used protease inhibitors can be studied for various serotypes of HIV-2 protease both in enzymatic and cell culture assays. In our experiments, optimization of the expression protocol led to a relatively stable enzyme, for cell culture assays, the efficiency of transfection and transduction capability of the modified vector was tested and was not found to differ from that of the wild-type, moreover, a 2nd generation protease inhibitor was used to demonstrate the usefulness of the system. The combination of assays performed with our cassette system is expected to provide an accurate measure of the efficacy of currently used; as well as experimental protease inhibitors on HIV-2.

Anticholesterol antibodies (ACHA) are natural antibodies against the 3beta-OH group of cholesterol. Since lipid disorders are common in HIV infection and HAART may further enhance dislipidaemia, we determined by using an ELISA method serum ACHA concentrations in HIV patients and healthy HIV-seronegative controls. ACHA levels were almost 4 times higher in the sera of 46 patients than in 110 controls. No difference in the specificity of ACHA was found between HIV-seropositive and HIV-seronegative sera. Binding of ACHA to cholesterol-coated plates from a HIV-seropositive serum was dose-dependently inhibited by preincubation with HIV-1(BA-L) preparation. Serum concentration of ACHA was significantly higher in the patients with low serum cholesterol levels than in those with normal cholesterol levels. HAART induced a marked drop of ACHA concentration. We found a significant negative correlation between the length of HAART and the ACHA levels. By contrast, HAART did not significantly influence total IgG concentration and titers of antibodies against 60 kD heat shock protein. Our findings indicate that high levels of ACHA in HIV-infection may contribute to the development of hypocholesterolaemia frequently observed in this disease.

Using the single-strand conformation polymorphism and heteroduplex analyses, the P53 and RB genes were analyzed in cell samples from twenty-eight patients with chronic myeloid leukemia (CML) both at diagnosis and at the onset of accelerated phase (AP) of the disease. No alterations of the P53 or RB genes were found in any of the chronic phase (CP) samples. Structural abnormalities of the P53 gene were observed in ten of twenty-eight AP samples within exons 4, 5, 7 and 9. Of the ten cases of AP disease with altered P53 genes, five patients also suffered from the deletion of the other allele. Alterations of the RB gene could be detected in six AP samples, and aberrant band patterns were found in the analysis of exons 2, 3, 4, 6, 7, 13, 14, 17, 21 and 26. Among the six AP samples with structural abnormalities of the RB gene, two showed the loss of the other allele. It is of note that alterations of both P53 and RB genes were observed in two AP samples. Our data strongly suggest that abnormalities of the P53 and RB genes and acceleration of CML are linked events in some cases of AP.

In humans the HIV infection results in a chronic disease with a permanent fight between factors controlling HIV and the escape of the virus. Fromthese control mechanisms the present review summarizes the role betwen complement and autoantibodies; the competition of complement and anti-HIV antibodies for binding sites, the role of mannan-binding lectin in the susceptibility to and in the survival after HIV infection, the contribution of complement-dependent enhancing type antibodies to the clinical progression of HIV disease as well as the changing pattern of some autoantibodies (mimicking MHC class II molecules, anti-heat shock protein 60 antibodies and anti-C1q antibodies) which were found to correlate to immunological and clinical parameters.

Human trophoblast populations from first-and third-trimester placentas produce interferons (IFNs) in the presence of growth factors (CSF and PDGF) or when infected with virus. The highly invasive extravillous trophoblast population produced a higher level of IFNs (three- to eightfold, P0.05) than the noninvasive villous trophoblast population when stimulated with growth factors and/or virus. The level of IFN produced was dependent on the type of trophoblast population, the type of inducer and the stage of differentiation of the trophoblasts. Tandem immunoaffinity chromatography of the virus-induced trophoblast IFNs resulted in the isolation of trophoblast IFN-alpha and -beta types. The purified trophoblast IFNs have antiviral, antiproliferative and immunoregulatory properties. Furthermore, the trophoblast IFNs inhibited the expression of proto-oncogenes such as EGF-R, c-erbB2 and c-fms reported to be involved in normal trophoblast growth and differentiation. These data suggest essential roles of interferons in normal human development during pregnancy.

The syncytiotrophoblast layer of the human placenta has an important role in limiting transplacental viral spread from mother to fetus. Human cytomegalovirus (HCMV) is capable of establishing a latent infection in syncytiotrophoblast cells, with restriction of gene expression to immediate-early and early proteins. We analyzed the extent of replication of human T cell leukemia-lymphoma virus type I (HTLV-I) in human term syncytiotrophoblasts infected with HTLV-I alone or coinfected with HTLV-I and HCMV. Although syncytiotrophoblasts could be infected with cell-free HTLV-I, no viral protein expression was found in the singly infected cells. On the contrary, coinfection of the cells with HTLV-I and HCMV resulted in simultaneous replication of both viruses. Bidirectional enhancing activities between HTLV-I and HCMV were mediated primarily by the Tax and immediate-early proteins, respectively. The stimulatory effect of HTLV-I Tax on HCMV replication appeared to be mediated partly by tumor necrosis factor beta and transforming growth factor beta-1. We observed formation of pseudotypes with HTLV-I nucleocapsids within HCMV envelopes, whereas HCMV was not pseudotyped by HTLV-I envelopes in dually infected syncytiotrophoblast cells. Our data suggest that in vivo dual infection of syncytiotrophoblast cells with HTLV-I and HCMV may facilitate the transplacental transmission of both viruses.

Two types of antibodies which previously were found to be inversely associated with CD4 + cell counts and which may contribute to the progression of HIV disease were measured in parallel in 55 serum samples of 7 longitudinally tested HIV-infected patients (4 homosexual men, 3 haemophilic men) and in 15 serum samples from 15 patients with advanced AIDS. HIV-infection enhancing antibodies were determined in the presence of near-physiologic human complement concentration using a complement receptor type 2 (CR2) carrying HIV-target cell line. IgG and IgA class autoantibodies directed against human IgG-Fab fragments were measured in specific ELISA assays. In agreement with our previous studies obtained in HIV-seropositive haemophilic patients, significant negative correlations were found between CD4 + cell counts and IgG anti-Fab and IgA anti-Fab antibodies (Spearman correlation coefficient r =−0.587, P r =−0.269, P =0.024, respectively). A significant positive correlation was observed between complement-dependent enhancing antibodies and IgA anti-Fab antibodies ( r =0.408, P =0.003), whereas the correlation with IgG anti-Fab antibodies was only weak ( r =0.288, P =0.034). Serum samples with high titres of complement-dependent enhancing antibodies had almost 3 times higher IgA anti-Fab autoantibody activity than sera with low titres ( P =0.0038). Our findings indicate that the two disease markers in HIV disease, enhancing antibodies and autoantibodies directed against the Fab moiety of IgG, are not identical. However, anti-Fab antibodies may contribute to complement-dependent HIV infection enhancement.

Summary Most studies to date indicate that the most frequent mode of vertical infection of HIV is transplacentally after hematogenous infection. Since virus is not reaching the fetus in about 70 per cent of the cases a strong defense system may exist. Based upon macroscopic and light microscopic examinations of the human placenta, responses to HIV infection are minimal however, the presence of the virus in most cases can be demonstrated in fetal placental monocytes (Hofbauer cells) and oftern in the trophoblastic cells. Virus presence in other cell types is reported less frequently. Infection of the trophoblastic cells appears possible both with free virus attaching to CD4 receptors, via virus-antibody complexes, and via maternal leukocytes attaching to trophoblastic cells. In vitro evidence further suggests that trophoblastic cells may both secrete free virus and deliver to CD4 positive lymphocytes. Trophoblast infection with HIV seems to be non-lytic but productive and the penetration further into the fetal tissue likely occurs via infection of Hofbauer cells. The trophoblastic cells are capable of producing both alpha, beta, and gamma interferon, in particular, during the first trimester. Their role(s) in placental response to HIV is under study. Maternal antibodies to HIV may enhance trophoblastic cell uptake of HIV. Furthermore, the maternal cellular cytotoxic immunune response to infected trophoblastic cells seems muted by unknown factor(s). Cytokines may play a central role in placental responses to HIV and external modification of placental defenses should include attempts to modify transmission to the fetus during pregnancy.

Summary Congenital vaccinia is a well known cause of fetal wastage. To investigate a possible mechanism of virus transmission from mother to fetus, human trophoblast isolated from 7–12 weeks placentae was challenged with vaccinia virus. First trimester trophoblast was susceptible to vaccinia virus infection in vitro , as determined by morphologic alterations, cessation of hCG synthesis, and production of progeny virus. A recombinant virus carrying HIV DNA sequences induced trophoblast expression of gp 160 and replicated to the same extent as the control wild-type strain. The results may have a bearing on the general pathogenesis of fetal infections, and serve as a reminder of the small but not negligible risk of vaccination with live virus in pregnancy.

The human placental trophoblasts which constitute the first fetal cells and form the major cell layer of the feto-maternal interface are potent producers of interferons (IFNs). The IFN production is dependent on the gestational age of the trophoblast, type of inducer and the stage of differentiation of the trophoblasts. First trimester trophoblast populations produce higher levels (5-6 times) of IFN than the third trimester trophoblasts when stimulated with viruses. Non-viral inducers, such as poly(rl).poly(rC), induce exclusively IFN-β whereas viruses such as Sendai and Newcastle Disease Virus (NDV) induce mixtures of IFN-α subtypes and IFN-β. Differentiation of mononuclear cytotrophoblasts into syncytiotrophoblasts in vitro increase the IFN production. High-performance and immunoaffinity chromatography of the virusinduced trophoblast IFN preparations resulted in the isolation of three antigenically distinct IFNs, namely, αI, αII1 (ω1), and β with molecular masses of 16, 22 and 24 kDa, respectively, on SDS-PAGE. The human trophoblast IFNs have physical and antiviral activities characteristic of the Type 1 IFNs. The possible roles of the trophoblast IFNs in human placental and fetal development are also discussed in this review.

CD8+ and CD8- subsets of peripheral blood natural killer (NK) cells were examined for susceptibility to infection with human immunodeficiency virus type 1 (HIV-1) and for the ability to produce various types of interferon (IFN) and tumor necrosis factor (TNF). HIV-1 was preferentially grown in CD8+ NK cells. The ability of CD8- NK cells to suppress HIV-1 replication was related to their ability to produce alpha IFN (IFN-alpha) upon viral induction. Induction with interleukin-2 resulted in IFN-gamma production in both subsets of NK cells. In the CD8+ subset, IFN-gamma and HIV-1 mutually enhanced the production of TNF alpha, leading to hyperactivation of viral replication, whereas in CD8- NK cells IFN-gamma primed HIV-induced IFN-alpha production. The dichotomous effects of IFN-gamma on HIV-1 replication were dependent on the IFN-alpha-producing ability of the cellular targets. These findings can explain the selective depletion of the CD16+ CD8+ subset that begins early in the in vivo HIV-1 infection.

Stimulation of human placental first and third trimester trophoblast and syncytiotrophoblast cultures with viruses [Newcastle Disease Virus (NDV) and Sendai virus] led to a high interferon (IFN) production. The magnitude of the production was dependent on the gestational age of the trophoblast, type of inducer and the stage of differentiation of the trophoblast. The data obtained indicated that the first trimester trophoblast cultures produced five to sixfold more IFN than the third trimester trophoblast on per cell basis whereas syncytiotrophoblast at term produced twice as much IFN than the mononuclear term trophoblast when stimulated with the viruses. NDV and Sendai virus produced different levels and composition of IFN-alpha and -beta in both first and third trimester trophoblast and syncytiotrophoblast cultures. Purification of the virus-induced trophoblast interferons (tro-IFNs) by tandem high-performance affinity chromatography resulted in specific activities between 0.7 and 2.7 x 10(8) IU/mg of protein when assayed on human amniotic WISH cells. The tro-IFN-alpha protected both human and bovine MDBK cells from virus infection whereas the tro-IFN-beta protected only the human cell lines tested. The possible roles of the tro-IFNs are discussed in light of the observed differences in trophoblast IFN response.

DNA sequences distantly related to the proviral DNA of HTLV-I were found in the leukemic cells of a Hungarian patient suffering from Sezary syndrome. Serum samples from the patient contained antibodies reactive with the internal core polypeptides of HTLV-1 and HTLV-I 1, but not with the env gene encoded type-specific HTLV antigens. The husband and daughter of the patient also had antibodies of the same specificity. These findings suggest the presence of a virus distantly related to HTLV-I and HTLV-II.

Human term-placental trophoblasts in primary culture were studied for an interferon (IFN) response when challenged with Sendai virus and compared to three choriocarcinoma cell lines, placental fibroblasts and placental macrophages. Normal trophoblasts were high producers and released both IFN-alpha and IFN-beta. In contrast, one choriocarcinoma cell line was a low producer and all malignant lines produced only IFN-beta. Circulating monocytes produce IFN-alpha but placental macrophages secreted IFN-beta and some IFN-alpha, suggesting that IFN production may be dependent on the stage of differentiation. A role for trophoblast IFNs in protection of the foetus against virus infections is proposed.

Human trophoblast cultures were established from term placentae. Upon exposure to polyriboinosinic-polyribocytidylic acid (poly(I:C)) the cultures produced interferon. Results of neutralization experiments and indirect immunofluorescence studies indicated that the trophoblast produces beta-interferon. The fusion of trophoblast cells into syncytia was accompanied by a restriction in interferon release after stimulation with poly(I:C). It was also demonstrated that the malignant choriocarcinoma cell line JAR produced less interferon than the non-transformed cyto- or syncytiotrophoblast.

The phenotypic mixing between human immunodeficiency virus type 1 (HIV-1) and vesicular stomatitis virus (VSV) has been exploited to assay the susceptibility of human term syncytiotrophoblast cells to penetration by various strains of HIV-1. VSV (HIV-1IIIB) and VSV (HIV-1Ba-L) pseudotypes were found to enter syncytiotrophoblasts. Infection of syncytiotrophoblasts was mediated by envelope glycoproteins of IIIB and Ba-L strains of HIV-1. Although certain strains of HIV-1 could enter syncytiotrophoblasts, the cells did not exhibit permissiveness for HIV-1. The next studies tested the possibility that placental macrophages might induce replication of HIV-1 carried in syncytiotrophoblast cells and that infected syncytiotrophoblasts would be capable of transmitting virus into neighbouring macrophages. For this purpose, the macrophage-tropic Ba-L strain of HIV-1 was used. Interactions between syncytiotrophoblasts and macrophages activated HIV-1 from latency in syncytiotrophoblast cells, which delivered HIV-1 to cocultured macrophages. The stimulatory effect of coculture on HIV-1 gene expression was mediated by marked tumor necrosis factor-α and interleukin-6 release from macrophages, an effect caused by contact between the different placental cells. Results suggest an interactive role for the syncytiotrophoblast layer and placental macrophages in the dissemination of HIV-1 among placental tissue.

Serum samples of 589 haemodialysis patients were screened for HIV antibody by ELISA methods. Of these, 36 samples were found to be repeatedly reactive. None of the 36, however, could be confirmed by competitive enzyme immunoassays and Western blot; therefore, they were considered to be false positive. The sera could be divided in two groups. The sera of Group 1 were designated as the usual type of false positivity, caused most probably by anti-lymphocyte antibodies. In 19 sera, however, a special type of false positivity was found. These sera reacted strongly with the plates coated with the supernatants of HIV-infected cells but not with those of uninfected H9 cells. Three and two sera showed, respectively, positive immunofluorescence reaction with the HIV-infected, but not with the uninfected, H9 and CEM cells. Reactivity to HIV-infected H9 cells could be adsorbed from a part of these samples with lesser amounts of HIV-infected than uninfected H9 cells. This special type of false positivity was observed frequently (7/65) in patients who rejected a kidney graft. These findings suggest that this type of anti-HIV false positivity is due to antibodies reacting with cellular antigens present in HIV-infected but not in uninfected lymphocytes. Their appearance seems to be associated with the immunological activation occurring at graft rejection.

Epstein – Barr virus (EBV) and human immunodeficiency virus type 1 (HIV-1), as well as human T-cell leukemia-lymphoma virus type I (HTLV-I), may interact in the pathogenesis of human retroviral infections. The placental syncytiotrophoblast layer represents a barrier protecting the fetal compartment from exposure to retroviruses. We studied the interactions of EBV with HIV-1 and HTLV-I in human term syncytiotrophoblast cells to investigate the significance of double infections in transplacental transmission of human retroviruses. We found that syncytiotrophoblast cells could be productively infected with EBV. Dual infection of the cells with EBV and HTLV-I resulted in full replication cycle of otherwise latent HTLV-I. In contrast, the restricted permissiveness of syncytiotrophoblasts for HIV-1 was not influenced by coinfection of the cells with EBV. Infection of syncytiotrophoblast cells with EBV, but not HTLV-I, induced interleukin-2 and interleukin-6 secretion, and augmented secretion occurred on coinfection with both viruses. Coinfection of syncytiotrophoblast cells with EBV and HTLVI induced tumor necrosis factor-b and transforming growth factor-b1 secretion, but infection with either virus alone did not lead to secretion of these cytokines. Permissive replication cycle of HTLV-I was induced by the EBV immediate-early gene product Zta. Pseudotype formation between EBV and HTLV-I in coinfected syncytiotrophoblast cells was not found. Our data suggest that activation of HTLV-I gene expression by EBV in coinfected syncytiotrophoblast cells may be a mechanism for transplacental transmission of HTLV-I. q 1997 Academic Press

The development of erythroleukemia in Balb/c mice injected with Rauscher leukemia virus has been followed by indirect immunofluorescence technique and flow cytometry, using antiserum against disrupted of virions. The progression of the disease was accompanied by a great increase in the number of large, immunofluorescence positive cells. A subpopulation of normal spleen cells was also highly positive. The expression of the antigens in normal cells was examined in relation to the cell cycle. The majority of the S-G2/M phase cells were found in the antigen positive compartment of larger cells. A two-color analysis of immunofluorescence and DNA content revealed that the distribution of antigen expression in G1 phase was broad, gradually decreasing from a low-intensity mode. The cell with double DNA content distributed evenly around a modus of five-fold higher intensity. In experiments with stimulated bone-marrow cells, superiority of S-phase cells in anti-Rauscher serum binding was found. Cell-surface gp70 antigen is suggested to be involved in this cell-cycle dependent binding of antibodies by normal cells.