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CCL19 chemokine plays an important role in regulating DC traffic and recruitment of naïve T cells to the vicinity of activated DCs and macrophages. We have analyzed the regulation of CCL19 gene expression in human monocyte-derived DCs/macrophages infected with Salmonella enterica or Sendai virus. Computer analysis of the CCL19 promoter identified two putative NF-κB binding sites and one interferon stimulated response element (ISRE). Transcription factor binding experiments demonstrated that Salmonella or Sendai virus infection increased the binding of NF-κB proteins (p50, p52, p65, and RelB) to both CCL19 promoter NF-κB elements. Salmonella or Sendai virus infection also increased the binding of multiple IRFs, as well as STAT1 and STAT2, to the ISRE site. The role of NF-κB and various IRF family members in transcriptional control of CCL19 gene was further studied in transfection experiments. The expression of NF-κB dimers readily activated CCL19 promoter. The expression of IRF1, IRF3, or IRF7 proteins was also able to activate the promoter in the presence of Sendai virus infection. CCL19 promoter constructs containing mutated NF-κB and/or ISRE sites were weakly activated. Our data also suggests that c-Jun and c-Fos proteins binding to target AP-1 elements in CCL19 promoter plays a role in transcriptional control of CCL19. Further work focuses on the regulation of CCL19 in human macrophages and myeloid DCs.

Background: Hepatitis C virus (HCV) encodes several proteins that interfere with the host cell antiviral response. Previously, the serine protease NS3/4A was shown to inhibit IFN-β gene expression by blocking dsRNA-activated retinoic acid-inducible gene I (RIG-I) and Toll-like receptor 3 (TLR3)-mediated signaling pathways. Results: In the present work, we systematically studied the effect of all HCV proteins on IFN gene expression. NS2 and NS3/4A inhibited IFN gene activation. NS3/4A inhibited the Sendai virusinduced expression of multiple IFN (IFN-α, IFN-β and IFN-λ1/IL-29) and chemokine (CCL5, CXCL8 and CXCL10) gene promoters. NS2 and NS3/4A, but not its proteolytically inactive form NS3/4A-S139A, were found to inhibit promoter activity induced by RIG-I or its adaptor protein Cardif (or IPS-1/MAVS/VISA). Both endogenous and transfected Cardif were proteolytically cleaved by NS3/4A but not by NS2 indicating different mechanisms of inhibition of host cell cytokine production by these HCV encoded proteases. Cardif also strongly colocalized with NS3/ 4A at the mitochondrial membrane, implicating the mitochondrial membrane as the site for proteolytic cleavage. In many experimental systems, IFN priming dramatically enhances RNA virusinduced IFN gene expression; pretreatment of HEK293 cells with IFN-α strongly enhanced RIG-I expression, but failed to protect Cardif from NS3/4A-mediated cleavage and failed to restore Sendai virus-induced IFN-β gene expression. Conclusion: HCV NS2 and NS3/4A proteins were identified as potent inhibitors of cytokine gene expression suggesting an important role for HCV proteases in counteracting host cell antiviral response.

The first line of defense that gene delivery vectors, viral or non-viral, confront in cells is the type I interferon (IFN) response. Cells produce type I IFNs as a response to dsRNA (intermediates of viral replication) and other viral components. IFNs induce the expression of IFN stimulated genes that can be used as markers for IFN response. One of these IFN stimulated genes is the Myxovirus resistance protein A (MxA).

Rapid viral diagnosis was attempted in 106 military conscripts with pneumonia and in 101 military conscripts with other types of respiratory infections. Nasopharyngeal suction specimens (NPS) were assayed for viral antigens by immunofluorescence and enzyme immunoassay (EIA). Sputum specimens from 97 pneumonia patients were assayed for viral antigens by EIA. Also, 71 NPS and 13 sputum specimens were examined for the presence of adenovirus DNA by a sandwich hybridization (HYB) method. The reference test was adenovirus isolation in cell culture from the NPS. Adenoviruses were isolated from 6 pneumonia patients and from 20 patients with other respiratory infections. Of these 26 NPS, rapid diagnosis was successful in 13, 16, and 14 cases by EIA, immunofluorescence, and HYB, respectively. Four antigen-positive specimens were found among the 181 specimens which were negative by virus isolation. Sputum was found to contain adenovirus antigen by EIA in 5 of 97 tested specimens. Of these 97 specimens, 13 were selectively tested in HYB, and a positive signal was observed in 4 cases. Serological testing of paired sera revealed 23 adenovirus infections in the pneumonia group and 42 in the group with other respiratory infections. Other viral infections were found only sporadically. All rapid virus detection methods showed excellent specificity but had a lower sensitivity (60%) than virus isolation. Our results show that rapid methods for diagnosing respiratory adenovirus infections can be successfully used in selected groups of adults.

A simple method was developed for separation of cells from nasopharyngeal secretion for the diagnosis of respiratory virus infections by immunofluorescence microscopy. The diluted specimen, containing dithiothreitol to break up the mucus, was centrifuged once through a cushion of 20% Percoll (colloidal silica, a density gradient medium), which permitted sedimentation of cells through the cushion, but retained mucus on top of it. The pelleted cells were resuspended, and microscope slides were then prepared by standard techniques. The Percoll centrifugation method was also applicable for sputum and bronchoaveolar lavation specimens. Immunofluorescent antibody staining of nasopharyngeal secretions prepared by the described method was more sensitive than enzyme immunoassay for the detection of respiratory syncytial virus.

Culture, enzyme immunoassay (Chlamydiazyme™) and nucleic acid sandwich hybridization were compared in detecting Chlamydia trachomatis in uncomplicated genital tract infections. Urethral and cervical specimens were collected from 100 males and 100 females attending a sexually transmitted disease clinic. Chlamydial culture was performed under optimal conditions (duplicate inoculation within the day specimen was collected, culture in vials, monoclonal antibody staining of inclusions, blind passage for negative samples). Here the sensitivity of culture exceeded that of the rapid methods. The sensitivity of a chlamydial antigen detection method (Chlamydiazyme™) was 68% in male and 86% in female specimens, when compared with culture, and the specificity was 100% and 97%, respectively. Acinetobacter calcoace9icus present in clinical specimens did not interfere with Chlamydiazyme™. The sensitivity of the nucleic acid sandwich hybridization was 53% of that of the culture, and specificity 100%. By comparing the three methods it was apparent that the rapid methods did not reveal chlamydial infections not detectable by culture. Thus, if performed carefully, culture is the most sensitive diagnostic method in acute genital infections due to C. trachomatis.

The efficacy of enzyme immunoassay (EIA) in detecting diagnostic antibody rises to influenza A and B viruses was compared with complement fixation (CF) and hemagglutination inhibition (HI) tests in 455 patients with an acute respiratory infection. EIA and HI detected significantly more diagnostic antibody rises against influenza A than the CF method (96 and 87 vs. 47, respectively). In the case of influenza B significantly more diagnostic influenza B antibody rises were observed by EIA than by CF or HI (59 vs. 37 and 40, respectively). In most of the cases antibody rises in EIA were found in both IgG and IgA isotypes whereas increases in IgM antibodies were seen less frequently. Purified hemagglutinins (HA) were prepared from influenza A HI- and H3-subtypes and from influenza B viruses and used as antigens in EIA and the results were compared with those of HI. Infections caused by influenza A HI-subtype showed good homologous antibody responses in EIA but heterologous antibody responses to H3-subtype and influenza B HAs were frequently observed. Heterologous responses were clearly less frequent in patients with infections caused by the H3-subtype. Influenza B infections occasionally raised HA antibodies against influenza A H1-subtype but not to the H3-subtype. Interestingly, HI detected these heterologous responses at least as frequently as EIA. When whole viruses were used as antigens in EIA, subtype specificity was not observed and cross-reactions between influenza A and B virus antibodies were found. These observations suggest that, although EIA can show greater diagnostic efficacy over HI and CF methods, HI is still the serological method of choice in determining the causative subtype of influenza A virus infection.

A study of 690 osteoarthrosis (OA) patients with corresponding controls, and 475 soft-tissue rheumatism (STR) patients with the same number of controls was made in Finnish health centres. The aim of the study was to clarify certain etiological, social and therapeutical aspects. No clear differences were found in the working conditions of patients with OA, STR, or their controls, or in their occupational classes. OA patients were less satisfied with their working conditions than were the other. OA and STR patients had been more actively engaged in sports earlier in life, but during the study there were no differences; nor did the localisation of OA differ. The weight index qas slightly higher in OA, particularly when knee and ankle were affected, but not with arthrosis of the hip. There were no clear associations with other diseases. The maximum duration of a treatment period in this study was 1 month. The average sick leave in OA was 17.8 days, in STR 13.4 and in controls 15.4 days. After treatment, 31% of OA patients, and 56% of STR were recommended to resume work; 4% were recommended to change their occupation; 21% of OA and 7% of STR patients were recommended to retire on pension. Sulindac, indomethacin and many other drug combinations were used for medication. Certain side effects were found in about 5% of sulindac- and in 8-10% of indomethacin-treated patients. In 17% of OA and in 12% of STR patients, it was found necessary to consult a specialist for diagnosis or treatment.

Epithelial cells of the respiratory tract are the primary targets of measles virus (MV) infection. In this work we have studied the effect of MV infection on the activation of transcription factors nuclear factor (NF)-kappa B and signal transducer and activator of transcription (STAT) and the production of cytokines in the lung epithelial A549 cell line. NF-kappa B and STAT activation were induced by MV in A549 cells as analyzed by electrophoretic mobility shift assay. NF-kappa B activation was rapid and it was not inhibited by the protein synthesis inhibitor cycloheximide, suggesting that MV directly activates NF-kappa B. In contrast, Stat1, Stat3, and interferon-stimulated gene factor 3 (ISGF3) DNA binding was induced by MV infection with delayed kinetics compared to NF-kappa B activation. MV infection also resulted in an efficient interferon (IFN)-alpha/beta and interleukin-6 production. Cycloheximide and neutralizing anti-IFN-alpha/beta antibodies inhibited MV-induced activation of Stat1, Stat3, and ISGF3 DNA binding in A549 cells. In conclusion, the results suggest that MV infection activates transcription factors involved in the initiation of innate immune responses in epithelial cells by two different mechanisms: directly by leading to NF-kappa B activation and indirectly via IFN-alpha/beta leading to STAT activation.

Hepatitis B, C, and D viruses can infect liver cells and in some individuals establish a chronic phase of infection. Presently, relatively little information is available on the antiviral mechanisms in liver cells. Because no good in vitro model infection systems for hepatitis viruses are available, we have used influenza A, Sendai, and vesicular stomatitis (VSV) viruses to characterize interferon (IFN) responses and IFN-induced antiviral mechanisms in human hepatoma cell lines. HepG2 or HuH7 cells did not show any detectable IFN-alpha/beta production in response to influenza A or Sendai virus infections. Treatment of cells with IFN-alpha resulted in upregulation of IFN-alpha-inducible Mx, 2',5'-oligoadenylate synthetase (OAS) and HLA class I gene expression but only with exceptionally high levels of IFN-alpha (>/=100 IU/ml). Accordingly, high pretreatment levels of IFN-alpha, 1000 IU/ml for influenza A and VSV and 100 IU/ml for Sendai virus, were required before any detectable antiviral activity against these viruses was seen. IFN-gamma had some antiviral effect against influenza A virus but appeared to be ineffective against VSV and Sendai virus. IFN-gamma upregulated HLA class I protein expression, whereas Mx or OAS expression levels were not increased. There was a modest upregulation of HLA class I expression during Sendai virus infection, whereas influenza A virus infection resulted, after an initial weak upregulation, in a clear decrease in HLA class I expression at late times of infection. The results suggest that hepatoma cells may have intrinsically poor ability to produce and respond to type I IFNs, which may contribute to their inability to efficiently resist viral infections.

Fibronectin is a dimeric high-molecular-weight glycoprotein composed of two similar but not identical subunits. Differences have been observed between plasma and cell-derived fibronectins, and molecular and sequencing studies have revealed a domain structure. The characteristic features of fibronectin include its various molecular and biologic interactions, which can be assigned to the different domains of the molecule. Fibronectin was originally discovered as a protein missing from the surfaces of virus-transformed cells. The importance of functional oncogenes in the loss of extracellular matrices was later established in a number of studies using virus mutants that are temperature-sensitive for transformation. The loss of the extracellular matrix appears to be a result of multiple events in cells but is not a prerequisite for malignant growth in vivo. There appear to be multiple transformed phenotypes, and the loss of extracellular fibronectin may be due in part to proteolytic activity by the cells and other alterations caused by functioning oncogenes. The wide distribution of tissue and soluble forms of fibronectin offers multiple occasions for interactions with other molecules. Binding of fibronectin to viruses and purified viral proteins has been observed in a number of studies. Neuraminidase-sensitive virus-binding sites in the fibronectin molecule have tentatively been identified. Fibronectin also is capable of binding to various complement components, possibly making clearance of viral antigen-antibody-complement complexes, which are observed in the circulation during viral infection, more rapid. The virus-binding ability of fibronectin may play a role in modifying viral infections in the body.

To study the outcome of mumps virus encephalitis 47 patients were contacted 1-15 years after the acute encephalitis associated with mumps virus infection. Twenty-three patients experienced clinical sequelae such as difficulties in memory and learning, focal motor or sensory signs, and loss of hearing and visual acuity. Lumbar puncture was performed on 8 patients. Antibodies to mumps virus were detected in 6 cerebrospinal fluid (CSF) specimens using enzyme immunoassay and in 3 patients an abnormal serum/CSF antibody ratio was observed 11, 26 and 58 (controls greater than 85); 14.3, 1.4 and 6.1 years after the acute encephalitis, respectively. Antibodies to other microbes were either undetectable in the CSF or the serum/CSF ratios were normal. The clinical sequelae in about half of the patients and the signs of intrathecal mumps antibody production are suggestive of a chronic process in the central nervous system after encephalitis associated with mumps virus infection.

Immunoglobulin isotype composition of poliovirus antibodies was studied by isotype-specific solid-phase radio-immunoassay (RIA) in four patients with paralytic poliomyelitis, five adults receiving live poliovirus vaccine as a booster immunization, and seven children receiving first doses of inactivated poliovirus vaccine. In paralytic poliomyelitis serum and cerebrospinal flind (CSF) poliovirus antibodies were mainly of IgG1, IgG3, and IgA isotypes. IgM antibodies were found in sera but not in CSF. Either IgG2 and IgG4 antibodies were undetectable or the titers were low. In adults who had received live trivalent poliovirus vaccine, antibodies against poliovirus type 3 were detected in IgG1 (53% of total antibodies), IgG3 (25%), IgM (9%), IgA (8%), IgG2 (3%), and IgG4 (2%) isotypes. In prevaccination and late postvaccination sera the share of IgG3 antibodies was exceptionally high (35%). In children who received inactivated poliovirus vaccine, antibodies developed in IgG1 (53–61% of total antibodies for poliovirus types 1, 2, and 3), IgG3 (12–21%), and IgM (23–33%) isotypes. Antibody levels in IgG2, IgG4, and IgA isotypes were low and observed only in a few cases. Like other viral antibodies IgG1 and IgG3 isotypes were the major IgG subclasses in poliovirus antibodies.

The genome of influenza A viruses is composed of eight segments of negative polarity (see Chapter 1). In infected cells, these virion RNAs (vRNAs) are both transcribed into messenger RNAs (mRNAs) and replicated. The distinctive feature of influenza viral mRNA synthesis is that it is primed by 5′ capped (m7GpppNm-containing) fragments derived from newly synthesized host-cell RNA polymerase II transcripts (Bouloy et al., 1978, 1979; Krug et al.,1979; Plotch et al., 1979, 1981; Krug, 1981, 1983; Herz et al., 1981). The mRNA chains are elongated until a stretch of uridine residues is reached 17–22 nucleotides before the 5′ ends of the vRNAs, where transcription terminates and polyadenylate [poly(A)] is added to the mRNAs (Hay et al., 1977a; Robertson et al., 1981). For replication to occur, an alternative type of transcription is required that results in the production of full-length copies of the vRNAs. The full-length transcripts, or template RNAs, are initiated without a primer and are not terminated at the poly(A) site used during mRNA synthesis (Hay et al., 1977a, 1982). The second step in replication is the copying of the template RNAs into vRNAs. This synthesis also occurs without a primer, since the vRNAs contain 5′ triphosphorylated ends (Young and Content, 1971). The three types of virus-specific RNAs—mRNAs, template RNAs, and vRNAs— are all synthesized in the nucleus of infected cells (Herz et al., 1981; Jackson et al., 1982; Shapiro et al., 1987). During the early phase of infection, the synthesis of these three types of virus-specific RNAs is coupled (Hay et al., 1977a; G. L. Smith and Hay, 1982; Shapiro et al., 1987), whereas during the later phase of infection essentially only vRNAs are synthesized (Shapiro et al., 1987).

Sera from patients with acute hepatitis B, hepatitis B surface antigen (HBsAg)-positive chronic hepatitis, and from asymptomatic HBsAg carriers were evaluated by enzyme-linked immunosorbent assay and by platelet 125I-labeled staphylococcal protein A test for the presence of C3b-binding IgG, IgM, and IgA activity (immunoconglutinins and circulating immune complexes of IgG, IgM, and IgA classes), and for the presence of IgG binding onto platelets, respectively. Significantly elevated C3b-binding IgG, IgM, and IgA activity (P < 0.001) was found in patients with acute hepatitis B, C3b-binding IgG activity (P < 0.01) in chronic hepatitis patients, but normal C3b-binding Ig activity in HBsAg carriers and controls. Similarly, platelet-bound IgG was significantly elevated in patients with acute hepatitis B and HBsAg-positive chronic hepatitis (P < 0.001). Serial studies in acute hepatitis B demonstrated that C3b-binding IgG and IgA activity significantly correlated with serum aspartate transaminase and bilirubin (P < 0.01) and platelet-bound IgG with aspartate transaminase values (P < 0.05). In addition, a significant association of C3b-binding IgG activity with platelet-bound IgG was found (P < 0.01). The results suggest that C3b-binding ELISA and PIPA tests may have clinical and immunopathogenic importance in studies on hepatitis B infection.

Paired sera from 20 patients with acute mumps infection, 16 from persons vaccinated with live attenuated mumps virus vaccine, and 12 from persons vaccinated with formalin-inactivated virus vaccine were studied for mumps antibodies by single radial hemolysis (SRH), hemagglutination inhibition (HI), and by enzyme immunoassays (EIA) specific for whole virus, envelope glycoprotein, and nucleocapsid antibodies. Mumps patients had diagnostic rises in serum mumps antibodies in 90-100% of the cases depending on the method of assay. Vaccination resulted in seroconversion in 75-88% (live vaccine) and in 92% (inactivated vaccine) of the cases as detected by SRH or EIAs, whereas HI detected seroconversion only in 38% and 58% of the cases, respectively. Immunoprecipitation analyses revealed that all sera from mumps patients and nearly all postvaccination sera had antibodies against the main structural proteins of mumps virus. By immunoblotting, antibodies against denatured hemagglutinin-neuraminidase (HN) and fusion protein (F) were detected in 15-25% of mumps patients and persons vaccinated with live vaccine, whereas most postvaccination sera from those vaccinated with inactivated vaccine had HN (92%) and F (83%) protein antibodies, suggesting that antibodies against the denatured form of proteins are formed.

A sensitive enzyme immunoassay (EIA) for determining the biological activity of human interferon was developed. Green monkey kidney (Vero) cells and human embryonic lung (HEL) cells were grown in microtitre plates, treated with leukocyte interferon (IFN alpha) and infected with vesicular stomatitis virus (VSV). Cells were fixed with paraformaldehyde and permeabilized with Triton X-100. Viral antigen synthesis was measured by labelling the cells with VSV antiserum followed sequentially by protein A horseradish peroxidase conjugate and o-phenylenediamine. Interferon activity was detected as a lowering of the absorbance value from that of the virus control wells, reflecting the inhibition of virus protein synthesis by interferon. The minimum amount of interferon producing statistically significant (P less than 0.01) decrease of absorbance in Vero cells was 1-5 international units (I.U.)/ml as in the standard plaque reduction test the detection limit was 7.5 I.U./ml or more. In HEL cells the detection limit was 1 I.U./ml measured by EIA. The EIA for interferon activity is at least as sensitive as the traditional plaque reduction test. It is reproducible, easy to automatise and requires 7-10 times less cell culture materials than the plaque reduction test. We find it preferential especially when large numbers of specimens with limited volumes are to be analysed for interferon activity.

Paired sera from 784 patients with symptoms of acute respiratory disease were examined for antibodies against influenza A, B and parainfluenza (1 and 3) viruses by complement fixation (CF) and enzyme immunoassay (EIA). The internal variation of the EIA test results was low and an increase of 0.250 in absorbance values which corresponded to a two-fold increase in end-point titres was considered a diagnostic antibody rise. EIA detected significantly more diagnostic rises than the CF test in the case of influenza A (222 vs. 162, P less than 0.001) and parainfluenza virus antibodies (29 vs. 16, P less than 0.01). More diagnostic rises in influenza B antibodies were also observed by EIA compared to the CF test (104 vs. 99, not significant). There were only two patients who showed a diagnostic rise in CF antibodies (both influenza B) but not in EIA. Most often patients with a diagnostic antibody rise only by the EIA method had a two-fold rise in the respective CF antibodies (68% of cases). EIA was found to be a sensitive and reliable method for the serological diagnosis of influenza A, B and parainfluenza infections.

Normal human sera were tested for immune complexes (IC) by 3 different tests: Clq-binding ELISA (ClqB-ELISA), conglutinin binding ELISA (KgB-ELISA) and platelet iodinated protein A test (PIPA). 118 sera stored for various times were tested in 3 separate groups: 45 freshly obtained sera (group 1), 38 sera stored at -20°C for 1.5 years and frozen and thawed 5–8 times before testing (group 2), and 35 sera stored at -20°C for 4 years (group 3). Group 1 formed the ‘control’ group and test values exceeding the mean plus 3 S.D. of the controls were considered positive. By the ClqB-ELISA, 8% and 6% respectively of group 2 and 3 sera were positive. In the KgB-ELISA, the group 2 and 3 values were 37% and 29%, and in the PIPA test, 24% and 20%. The KgB-ELISA results correlated significantly ( r = 0.36, P P Special care in evaluating IC assay results should, therefore, be taken when stored sera are being tested for IC. Adequate controls of the same storage age should be included. High serum IgG concentrations may correlate with positive results in IC assays.

Complement fixation and enzyme immunoassay (EIA) were used for measuring viral antibodies in 103 military conscripts with pneumonia and 98 conscripts with other respiratory infections. Diagnostic rises in antibody to adenovirus were found in 23 (22%) patients with pneumonia and in 42 (43%) patients with other respiratory infections. EIA detected more diagnostic rises than did complement fixation (22 versus 17 for pneumonia and 42 versus 40 in other infections). Adenovirus antibodies were analyzed for different immunoglobulin classes by EIA. Diagnostic rises in immunoglobulin G (IgG) and IgA isotypes were observed in 89 and 77% of the cases, respectively. IgM antibodies were positive in 39% of the cases. In five cases (8%), the demonstration of adenovirus antibodies of the IgM class was the only serological evidence for adenovirus infection. The present study demonstrates that the immunoglobulin class-specific EIA is a sensitive method for the diagnosis of respiratory adenovirus infections.

Symptoms of severe encephalomyelitis developed in a 31-year-old man in 1967. He had a high serum antibody titre to mumps virus associated with a polymorphic cell reaction and an increased protein concentration in cerebrospinal fluid (CSF). He recovered considerably within a year and was able to resume work. In 1975 his condition deteriorated again; it improved during the following few years, but a further deterioration then occurred. In March, 1981, the complement-fixing antibody titre to mumps virus was 1/32 in the serum and 1/4 in the CSF. In November, 1981, the CSF IgG index was increased and the altered serum/CSF antibody ratio persisted. The specificity of the altered antibody ratio was confirmed by the single radial haemolysis test and an immunoassay specific for mumps virus. Antibodies against the mumps virus envelope glycoprotein, M-protein, and nucleoprotein could be demonstrated by immunoprecipitation and the antibody patterns in serum and CSF were similar. Antibodies against other microorganisms were not detected in the patient's CSF, and mumps antibodies were not found in the CSF specimens of 57 control patients. This case may be an example of a new disease—chronic mumps virus infection in the central nervous system.

Puumala virus (PUU) is a member of the genus Hantavirus in the family Bunyaviridae and the causative agent of nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome. Sera of nephropathia epidemica patients react specifically with PUU nucleocapsid (N) protein. In order to safely provide large quantities of antigen for diagnostic purposes, PUU Sotkamo strain N protein was expressed by using the baculovirus system in Sf9 insect cells to up to 30 to 50% of the total cellular protein. The recombinant N protein (bac-PUU-N) was solubilized with 6 M urea, dialyzed, and purified by anion-exchange liquid chromatography. In an immunoglobulin M mu-capture assay purified and unpurified bac-PUU-N antigen showed identical results compared with the results of a similar assay based on native PUU antigen grown in Vero E6 cells. An immunoglobulin G monoclonal antibody-capture assay based on unpurified bac-PUU-N also showed results identical to those of an assay with native PUU-N antigen. Moreover, a panel of monoclonal antibodies reactive with eight different epitopes showed identical reactivity patterns with both natural and bac-PUU-N antigen, while two epitopes in PUU-N expressed as a fusion protein in Escherichia coli were not recognized. Puumala hantavirus N protein expressed by the baculovirus system offers a safe and inexpensive source of specific antigen for large-scale diagnostic and seroepidemiological purposes.

All-trans-retinoic acid (ATRA) is the drug of choice in the treatment of acute promyelocytic leukemia (APL). ATRA induces both in vitro and in vivo differentiation of APL cells into mature granulocytes. However, the molecular mechanisms involved in ATRA-dependent growth inhibition and cellular differentiation are not presently understood. The NB4 cell line, which is derived from the bone marrow of a patient with APL during relapse, can be used as a model system to study the growth and differentiation of APL cells. Because interferon (IFN) regulatory factors (IRF-1 and IRF-2) and other IFN-inducible gene products regulate cell growth, we analyzed the effects of ATRA on the expression of these genes. We show that ATRA directly activates IRF-1 gene expression, followed by activation of IRF-2 and 2′–5′ oligoadenylate synthetase (OAS) gene expression with slower kinetics. In addition to NB4 cells, ATRA also activated IRF-1 gene expression in HL-60, U937, and THP-1 cells, which all respond to ATRA by growth inhibition. A more than additive increase in IRF-1 gene expression was seen with ATRA and IFN-gamma in NB4 cells. ATRA did not activate nuclear factor kappa B or signal transducer and activator of transcription (STAT) activation pathways, suggesting that an alternate mechanism is involved in IRF-1 gene activation. The ATRA-induced expression of IRF-1, an activator of transcription and repressor of transformation, may be one of the molecular mechanisms of ATRA-induced growth inhibition, and the basis for the synergistic actions of ATRA and IFNs in myeloid leukemia cells.

Puumala virus (PUU) is a member of the Hantavi rus genus in the family Bunyaviridae and the etiologic agent of nephropathia epidemica (NE), a form of haemorrhagic fever with renal syndrome (HFRS). In this study we compared the immunofluorescence patterns of NE sera and antibodies raised against recombinant PUU proteins and confirm that the nucleocapsid protein is the major target in the early IgG response of NE patients and provides the molecular basis for simple and rapid differentiation between acute illness and old immunity by granular vs. diffuse fluorescence staining in the indirect immunofluorescence test. The differential kinetics of B-cell responses to PUU nucleocapsid vs. envelope proteins was emphasized further by the end-point titres of IgG antibodies to N, G1 and G2 proteins in NE patients. The granular fluorescence correlated with low IgG avidity in 99.8%. and diffuse fluorescence with high avidity in 100% of 617 NE sera studied. Epitope scanning with overlapping 14-mer peptides covering the whole nucleocapsid protein by a shift of 3 amino acids revealed six major antigenic epitopes recognized by sera from acute-phase NE patients. The epitopes clustered mainly in the hydrophilic regions, and two of them in a highly variable region which could probably serve as an antigen to distinguish serologically between infections of closely related hantaviruses, some apparently apathogenic, some causing lethal infections. The anti-peptide epitope pattern varied between different individuals and a collection of several pinbound peptides was needed to be recognised by most NE sera studied. © 1995 Wiley-Liss, Inc.

We recently reported that dendritic cells (DC) infected with Mycobacterium tuberculosis (Mtb) produce Th1/IFN-γ-inducing cytokines, IFN-αβ and IL-12. In the present article, we show that maturing Mtb-infected DC express high levels of CCR7 and they become responsive to its ligand CCL21. Conversely, CCR5 expression was rapidly lost from the cell surface following Mtb infection. High levels of CCL3 and CCL4 were produced within 8 h after infection, which is likely to account for the observed CCR5 down-modulation on Mtb-infected DC. In addition, Mtb infection stimulated the secretion of CXCL9 and CXCL10. Interestingly, the synthesis of CXCL10 was mainly dependent on the Mtb-induced production of IFN-αβ. Indeed, IFN-αβ neutralization down-regulated CXCL10 expression, whereas the expression of CXCL9 appeared to be unaffected. The chemotactic activity of the Mtb-infected DC supernatants was evaluated by migration assays using activated NK, CD4+, and CD8+ cells that expressed both CCR5 and CXCR3. Mtb-induced expression of CCL3, CCL4, CXCL9, and CXCL10 was involved in the stimulation of NK and T cell migration. In accordance with the data on the IFN-αβ-induced expression of CXCL10, neutralization of IFN-αβ significantly reduced the chemotactic activity of the supernatant from Mtb-infected DC. This indicates that IFN-αβ may modulate the immune response through the expression of CXCL10, which along with CXCL9, CCL3, and CCL4 participates in the recruitment and selective homing of activated/effector cells, which are known to accumulate at the site of Mtb infection and take part in the formation of the granulomas.

Since May 2009, the pandemic influenza A(H1N1) virus has been spreading throughout the world. Epidemiological data indicate that the elderly are underrepresented among the ill individuals. Approximately 1,000 serum specimens collected in Finland in 2004 and 2005 from individuals born between 1909 and 2005, were analysed by haemagglutination-inhibition test for the presence of antibodies against the 2009 pandemic influenza A(H1N1) and recently circulating seasonal influenza A viruses. Ninety-six per cent of individuals born between 1909 and 1919 had antibodies against the 2009 pandemic influenza virus, while in age groups born between 1920 and 1944, the prevalence varied from 77% to 14%. Most individuals born after 1944 lacked antibodies to the pandemic virus. In sequence comparisons the haemagglutinin (HA) gene of the 2009 pandemic influenza A(H1N1) virus was closely related to that of the Spanish influenza and 1976 swine influenza viruses. Based on the three-dimensional structure of the HA molecule, the antigenic epitopes of the pandemic virus HA are more closely related to those of the Spanish influenza HA than to those of recent seasonal influenza A(H1N1) viruses. Among the elderly, cross-reactive antibodies against the 2009 pandemic influenza virus, which likely originate from infections caused by the Spanish influenza virus and its immediate descendants, may provide protective immunity against the present pandemic virus.

TLRs play a critical role in early innate immune response to virus infection. TLR3 together with TLR7 and TLR8 constitute a powerful system to detect genetic material of RNA viruses. TLR3 has been shown to bind viral dsRNA whereas TLR7 and TLR8 are receptors for viral single-stranded RNA. In this report we show that TLR7 or TLR8 are not expressed in human epithelial A549 cells or in HUVECs. Accordingly, A549 cells and HUVECs were unresponsive to TLR7/8 ligand R848. TLR3 was expressed at a higher level in HUVECs than in A549 cells. The TLR3 ligand poly(I:C) up-regulated IFN-β, IL-28, IL-29, STAT1, and TLR3 expression in HUVECs but not in A549 cells. An enhanced TLR3 expression by transfection or by IFN-α stimulation conferred poly(I:C) responsiveness in A549 cells. Similarly, IFN-α pretreatment strongly enhanced poly(I:C)-induced activation of IFN-β, IL-28, and IL-29 genes also in HUVECs. In conclusion, our results suggest that IFN-α-induced up-regulation of TLR3 expression is involved in dsRNA activated antiviral response in human epithelial and endothelial cells.

The cDNA for the developmentally regulated, neurite outgrowth-promoting protein HB-GAM (heparin-binding growth-associated molecule) was recently cloned and shown to encode a novel lysine-rich sequence that is homologous with retinoic acid-induced sequences suggested to function in cell differentiation (Merenmies, J., and Rauvala, H. (1990) J. Biol. Chem. 265, 16721-16724). The same sequence was found for the mitogenic and neurite outgrowth-promoting protein pleiotrophin (Li, Y.-S., Milner, P. G., Chauhan, A. K., Watson, M. A., Hoffman, R. M., Kodner, C. M., Milbrandt, J., and Deuel, T. F. (1990) Science 250, 1690-1694). In this study, we have constructed a recombinant baculovirus using the cDNA that encodes the putative preprotein of HB-GAM. The putative secretion signal of HB-GAM is cleaved off in the baculovirus expression system, and the recombinant protein is rapidly secreted to the culture medium. Recombinant HB-GAM purified from the culture medium retains the biochemical characteristics and the neurite outgrowth-promoting activity found for the tissue-derived protein. Studies on the neurite outgrowth-promoting activity suggest that HB-GAM functions as an extracellular matrix-associated protein that enhances axonal growth in perinatal cerebral neurons of the rat. Since the same predicted amino acid sequence has been ascribed to a mitogenic protein, mitogenic activities of the recombinant HB-GAM and of tissue-derived HB-GAM fractions were also studied. Recombinant HB-GAM did not display any significant mitogenic activity, suggesting that tissue-derived HB-GAM preparations may contain other heparin-binding mitogenic factors. We identified in brain-derived HB-GAM fractions a 17-kDa protein (p17) that is detached from heparin by a slightly higher salt concentration as compared to HB-GAM. We suggest that p17 is structurally distinct from HB-GAM and responsible for the mitogenic actions of tissue-derived HB-GAM fractions.