Mapping mutations in influenza A virus resistant to norakin.

To elucidate the mode of action of norakin against influenza A virus we sequenced the hemagglutinin gene of 11 norakin-resistant mutants. Resistance was coupled with 1-3 amino acid exchanges. The majority of mutations was localized in the HA2 polypeptide and was mostly associated with changes in charge or polarity of the amino acids. The amino acid substitutions are discussed in the context of the 3D structure of X31 hemagglutinin considered to be representative of the influenza hemagglutinins. Most of the mutations appear to destabilize the pH 7.0 structure by distorting or destroying hydrogen bonds as well as salt-bridges which are responsible for intra- and intersubunit contacts, while others destabilize the location of the fusion peptide, facilitating conformational changes in the presence of the inhibitor.

Catecholamines trigger IL-10 release in acute systemic stress reaction by direct stimulation of its promoter/enhancer activity in monocytic cells.

Acute stress reactions (e.g. linked with trauma, major surgery, psychic stress and myocardial infarction) are accompanied with temporary systemic release of the anti-inflammatory cytokine IL-10 followed by immunodepression. Since an association between activation of the sympathetic system and IL-10 release has been described, we studied the influence of catecholamines on its promoter activity in vitro. Using reporter gene assays we demonstrated that catecholamines in monocytic cells directly stimulate the IL-10 promoter/enhancer via a cAMP/protein kinase A-dependent pathway. A cAMP responsive element was identified as major target. Thus, catecholamines are directly involved in the regulation of immunoresponsiveness under stressful conditions.

Cytomegalovirus infection in transplant recipients. The role of tumor necrosis factor.

Human cytomegalovirus (CMV) infection is an important cause of morbidity and mortality in transplant recipients. CMV infection commonly results from the reactivation of a latent infection. Using a set of monoclonal anti-CMV antibodies, we found CMV antigen expression in peripheral blood mononuclear cells (PBMNC), particularly in monocytes, in 312 of 816 samples from 190 allograft recipients. The detection of CMV-IE antigens and CMV-IE DNA in PBMNC indicates that positive cells may represent truly infected cells. The relation between increased cytokine plasma levels (particularly following treatment by pan-T cell antibodies) and the appearance of CMV antigens in PBMNC suggests that cytokines may play an important role in the reversal of CMV latency. This hypothesis is supported by our finding that tumor necrosis factor-alpha (TNF) is able to stimulate the activity of the CMV-IE enhancer/promoter region in the human monocytic cell line, HL-60. The interleukins 1, 2, 3, 4, 6, 8 and 10; transforming growth factor-beta; interferongamma; and granulocyte/macrophage colony-stimulating factor did not show any enhancing effect on the CMV promoter activity. Thus, TNF-alpha seems to play a key role in regulating the balance between latency and reactivation of CMV infection. Inhibition of TNF-alpha release or action may be an alternative strategy for preventing CMV-associated morbidity in allograft recipients.

Molecular cloning of integrated proviral DNA of bovine leukaemia virus from virus-producing foetal lamb kidney cells.

Over 90% of the total viral information together with the adjacent 5' cellular sequences were cloned in the lambdoid spi selection vector lambda-2558 by taking advantage of the unique EcoRI restriction site very close to the 3' long terminal repeat. Of the fifteen isolated recombinant phages with viral inserts, one has been propagated and its DNA isolated and subjected to preliminary restriction endonuclease analysis. The proviral insert has been found to be almost identical to the unintegrated proviral DNA reported by Kashmiri et al. (1984).

Characterization of RD114-related endogenous cat retroviral elements ECE2.

RD114 virus is an endogenous xenotropic Type C retrovirus of domestic cat. Previously, it had been shown that genomic DNA of cat contained approximately 20 copies of RD114-related sequences. Only one encoded for the replication-competent RD114 virus. The endogenous sequences exhibited substantial sequence conservation within the gag and pol genes and LTRs, but were characterized by deletions and substitutions within the env region. The endogenous cat retroviral element ECE2 was isolated by screening a genomic DNA library of cat liver DNA with an env-specific cDNA of RD114 virus. It contained an env region which differed from all RD114-related sequences so far isolated and was homologous to the corresponding region of replication-competent RD114 with regard to their restriction map and partial sequence analysis (p20). Otherwise, ECE2 had a deletion of approximately 1 kbp in the putative pol gene and, therefore, did not represent the locus of inducible RD114 virus.

[Analysis of the serodiagnostic reactions of the bacterium Clostridium butyricum strain CNRZ 528 using passive hemagglutination]. / Untersuchungen zur Serodiagnostik von Stäbchen des Stammes Clostridium butyricum CNRZ 528 mit Hilfe der passiven Hämagglutination.

For serologic tumour diagnostics a specific proof of antibodies against rods of Clostridium butyricum CNRZ 528 by passive hemagglutination has been described. We were able to prepare an antigen from the cell extract (crude-antigen) of rods without flagella by gel filtration, that after fixation on the surface of human-erythrocytes, stabilized by tannin or glutaraldehyde, agglutinates specifically with rod-antibodies of rabbit-hyperimmunserum. Simultaneously however, there was no cross-reactivity with spore-antibodies of rabbit-hyperimmunserum. Sensitivity and specificity of this test are dependent on an optimal dose of antigen bound to the surface of the erythrocytes. The optimization of the assay has to be achieved by the testing of patients sera.

[Synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis]. / Synthese Poly(A)-haltiger RNA in auswachsenden Sporen von Bacillus subtilis.

The synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis was studied. A significant amount of RNA puls-labelled with 3H-uridine is polyadenylated. With the beginning of RNA synthesis in outgrowing spores labelled poly(A)-containing RNA was detected. The amount of poly(A)-RNA during the outgrowth and first cell division remains constant. Besides poly(A)-RNA the synthesis of tRNA and rRNA occurs. These results indicate a simultaneous activation of synthesis of tRNA, rRNA as well as of poly(A)-containing RNA during outgrowth of B. subtilis spores.

[Cell division and macromolecular synthesis in growing spores of a temperature sensitive filamentous mutant of Bacillus subtilis]. / Zellteilung und Makromolekülsynthesen in auswachsenden Sporen einer temperatursensitiven filamentösen Mutante von Bacillus subtilis.

A temperature sensitive mutant of Bacillus subtilis SB 19, strain ts 33-6 was characterized. This strain grows at 46 degrees C (restrictive temperature) with reduced intensity without septation processes. Under restrictive conditions DNA- and RNA-synthesis are remarkably reduced. DNA, however, is synthesized continuously under restrictive conditions causing the formation of multinuclear filaments. Septation, induced at permissive temperature, is not prevented under restrictive conditions. That means that under restrictive conditions initiation of septation is blocked whereas formation of septa can be observed. Shift-up experiments have shown that the initiation of septation processes occurs at an early stage of cell cycle.

Mechanisms of human cytomegalovirus (HCMV) (re)activation and its impact on organ transplant patients.

Human cytomegalovirus (HCMV) infection plays an important role in transplant patients. Its impact is both direct and indirect. This review focuses on new aspects of HCMV (re)activation and HCMV related pathology, particularly HCMV-associated renal allograft injury. During the last two years we have learned that HCMV is more frequently (re)activated, even in healthy people, than previously expected. Inflammatory as well as stress mediators and some drugs may (re)activate the virus by using distinct intracellular pathways. Commonly, HCMV (re)activation is accompanied by HCMV antigenemia/DNAemia, suggesting that precursor cells in the bone marrow play an important role as a reservoir of latent virus. However, local HCMV (re)activation (colon, lung) without detection of active HCMV infection in the peripheral blood is possible. In healthy people a sufficient type 1 T-cell response controls the active HCMV infection, while in patients with severe immune deficiency (AIDS, high-dose immunosuppression) the virus can spread in an uncontrolled fashion and induce 'classic' HCMV disease. In patients with moderate immune deficiency (e.g. long-term transplant patients on low-dose immunosuppression) virus spreading is controlled but the elimination of cells harboring the active virus may be insufficient. The resulting persistent HCMV antigenemia may induce chronic inflammatory processes leading to tissue injury, particularly in the allograft. Therefore, antiviral therapy may be useful in patients suffering from graft deterioration with otherwise clinically symptomless HCMV infection. HCMV-related immune deficiency with an increased risk of developing bacterial/fungal superinfections is frequently seen in patients with symptomatic HCMV disease but not in asymptomatic CMV antigenemia. The risk of developing superinfections can be predicted by flow-cytometric monitoring of peripheral blood monocytes.

Monitoring of patients for cytomegalovirus after organ transplantation by centrifugation culture and PCR.

A modified centrifugation culture technique and a polymerase chain reaction (PCR) is described for detection of early antigen and IE antigen DNA, respectively, for rapid and sensitive monitoring of active cytomegalovirus (HCMV) infection after organ transplantation. In a preliminary study, 541 clinical specimens (blood, urine, bronchoalveolar lavage, pharyngeal wash, sputum) from 59 organ recipients were assayed for HCMV antigen by centrifugation culture; 144 samples were tested by PCR simultaneously. Antigenemia detected by centrifugation culture correlated strongly with active HCMV infection and clinical symptoms and proved useful for monitoring the efficacy of antiviral therapy. PCR was more sensitive in an earlier phase of infection when centrifugation culture was still negative. The clinical usefulness of both methods is discussed.

Mutations in the hemagglutinin gene associated with influenza virus resistance to norakin.

The nucleotide sequences of the hemagglutinin genes of four norakin-resistant mutants of Influenza A/FPV/Weybridge were determined and compared to the wild-type hemagglutinin. All mutants show one or two amino acid substitutions which are discussed to destabilise the pH 7 conformation of hemagglutinin.

[Acute illness following admission to child day care centers and the use of antimicrobial agents]. / Das akute Krankheitsgeschehen nach Krippenaufnahme und der Einsatz von Antimikrobiotika.

1. During the first six months after the admission to day-care acute infections of children are very frequent. This gives rise to the question for the justification of the indication of antibacterial chemotherapy during this period. 2. During the mentioned period all acute illnesses and the corresponding therapies of total 361 children in Berlin, the capital of the GDR, Leipzig and Schwedt are analysed. 3. The local differences of the appearance of the antibacterial chemotherapy are not caused by the type and frequency of the diagnosis, but reflect the inadequate consideration for the therapy recommendations given by the Society of Pädiatrics. 4. The correlations between antibacterial chemotherapy and the appearance of acute respiration illnesses (ARI) suggest the influence of the bacteria on the origin of these illnesses of day-cared children.

Cloning of the resistant EcoRII recognition site of phage T7 into an EcoRII-sensitive plasmid makes the site susceptible to the restriction enzyme.

The recognition sequence 5'-CC(A/T)GG for EcoRII in the bacteriophage T7 genome is refractory to this restriction endonuclease, despite not bearing the specific (protective) methylation. Following the integration of this site as part of a 219 bp fragment (in which the recognition sequence is flanked by about 100 bp of T7 origin) into the EcoRII-sensitive vector pUC18, the T7 site becomes susceptible to cleavage, too. The same is true of recombinant pBR322 plasmids containing the T7-derived recognition site. The results show that the flanking sequences are not immediately responsible for the refractory behaviour of EcoRII sites and are in agreement with data according to which EcoRII requires the coordinated presence of at least two recognition sites in its DNA substrate.

Subtype H7 influenza viruses: comparative antigenic and molecular analysis of the HA-, M-, and NS-genes.

Antigenic analysis of the haemagglutinin and matrix protein with corresponding sets of monoclonal antibodies as well as sequence analysis of HA-, M-, and NS-genes were carried out to establish antigenic and genetic relationships between four fowl plague virus (FPV) strains of H7 subtype. The data obtained revealed close genetic relatedness between the oldest known influenza A virus, A/chicken/Brescia/1902 (H7N7), and two FPV strains, A/FPV/Dobson (H7N7) and A/FPV/Weybridge (H7N7). These three strains apparently differ in all genes investigated from the A/FPV/Rostock isolate.

Human cytomegalovirus reactivation in bone-marrow-derived granulocyte/monocyte progenitor cells and mature monocytes.

Monocyte/granulocyte progenitor cells of the bone marrow are a major site of human cytomegalovirus (HCMV) latency. The mechanisms of establishment and maintenance of HCMV latency are still unknown. Reactivation of the latent virus in bone-marrow-derived progenitor cells has been demonstrated in vitro and suggested to occur also in vivo. Clinical studies have shown that reactivation is a rather frequent event not only in immunosuppressed but also in nonimmunosuppressed patients and in healthy blood donors. At least three independent mechanisms of virus reactivation are discussed: systemic inflammation connected with strong tumor necrosis factor alpha release; application of cAMP-elevating drugs, and highly stressful events associated with increased plasma catecholamine levels.

CCAAT/enhancer-binding proteins alpha and beta negatively influence the capacity of tumor necrosis factor alpha to up-regulate the human cytomegalovirus IE1/2 enhancer/promoter by nuclear factor kappaB during monocyte differentiation.

Recently we demonstrated that the ability of tumor necrosis factor alpha (TNFalpha) to stimulate the human cytomegalovirus (HCMV) IE1/2 enhancer/promoter activity in myeloid progenitor-like cells decreases when these cells differentiate into promonocytic cells. In addition, TNFalpha stimulation in the progenitor-like cell line HL-60 was shown to be mediated by nuclear factor kappaB (NF-kappaB) activation and its binding to the 18-base pair sequence motifs of the IE1/2 enhancer. We demonstrate here that the cell differentiation-dependent reduction of TNFalpha stimulation is not due to insufficient NF-kappaB activation but correlates with increased synthesis of the monocyte differentiation-associated factors CCAAT/enhancer-binding protein (C/EBP) alpha and beta. Overexpression of C/EBPalpha/beta in HL-60 cells, which normally produce only very small amounts of C/EBP, stimulated the basal activity of the promoter in the absence of NF-kappaB but suppressed the stimulatory effect of TNFalpha. A novel C/EBP-binding site was identified in the IE1/2 enhancer directly downstream of a NF-kappaB site. In order to understand the mechanisms of interaction, we used an IE1/2 promoter mutant that failed to bind C/EBP at this position and several constructs that contained exclusively NF-kappaB- and/or C/EBP-binding sites upstream of the minimal IE1/2 promoter. We could demonstrate that C/EBPalpha/beta interacts with NF-kappaB p65 and displays inhibitory activity even in the absence of direct DNA binding by forming p65-C/EBP-containing protein complexes bound to the NF-kappaB site. Moreover, C/EBP binding to the DNA adjacent to NF-kappaB supports the down-regulatory effect of C/EBPs possibly due to stabilization of a multimeric NF-kappaB-C/EBP complex. Our results show that cell differentiation factors may interfere with TNFalpha-induced human cytomegalovirus gene (re)activation.

Effects of perfluorocarbons and perfluorocarbons/surfactant emulsions on growth and viability of group B streptococci and Escherichia coli.

OBJECTIVE: Partial liquid ventilation with perfluorocarbons (PFC) might be used as a new ventilatory strategy to treat respiratory insufficiency in congenital pneumonia. The present study investigates for the first time effects of PFC on growth and viability of group B streptococci (GBS) and Escherichia coli, bacteria frequently causing congenital pneumonia. DESIGN: Prospective, in vitro study. SETTING: Research laboratory in a university. MATERIAL: Group B streptococci 090 Ia HD Colindale and E. coli K12, JM 101. INTERVENTIONS: E. coli (10(7)/mL) were grown in the absence or presence of different PFC (RM 101, PF 5080, FO 6167) for up to 6 hrs. To study bacterial viability, GBS (5 x 10(7)/mL) were incubated in saline with or without different PFC, PFC/surfactant emulsions, or surfactant (Curosurf) for up to 5 hrs. Every 2 hrs, the colony forming units were determined by plating different dilutions of bacteria on agar. MEASUREMENTS AND MAIN RESULTS: RM 101 or PF 5080 alone and in emulsions with surfactant had no effect on viability of GBS or growth of E. coli. For FO 6167, a previously described toxicity was found, even if 1 mL of GBS suspension was incubated with only 100 microL of FO 6167, verifying the experimental design that guarantees a PFC bacteria contact. The toxic effects were almost prevented by forming a PFC-in-surfactant emulsion but not by preincubation of GBS with surfactant and subsequent FO 6167 exposure. CONCLUSION: RM 101 and PF 5080 did not influence bacterial growth in vitro; direct effects on bacterial proliferation during partial liquid ventilation in congenital pneumonia seem, therefore, unlikely. Interestingly, we found that the known toxic effects of FO 6167 can be prevented by covering PFC with a surfactant film. Surfactant reduced the cytotoxic effects of FO 6167, probably by preventing a direct contact between FO 6167 and the bacterial cell wall.

Inactivation of the very strong HCMV immediate early promoter by DNA CpG methylation in vitro.

The influence of DNA methylation in vitro on the activity of the very strong human cytomegalovirus (HCMV) major immediate early (IE) modulator/enhancer/promoter region was investigated by transient transfection experiments of premonocytic HL-60 cells. While sequence-specific methylation of the major IE enhancer and/or modulator with the cytosine methyl-transferases FnuDII, HhaI and HaeIII had no significant effect, the promoter activity was completely repressed by methylation of the cytosine in 5'-CpG sites with the Spiroplasma methyltransferase SssI. Addition of TNF-alpha or PMA which are strong stimulators of HCMV major IE enhancer/promoter activity in premonocytic HL-60 cells had no effect on repression. Inactivation of the IE enhancer/promoter via methylation by M.SssI could be partially alleviated by co-transfection with an excess of untranscribable highly methylated DNA. These results indicate that a methyl-CpG binding factor is involved as mediator in the inhibitory effect of HCMV enhancer/promoter methylation. Taken together, the HCMV major IE enhancer/ promoter has been shown to be susceptible to transcriptional inactivation by methylation of the cytosines in CpG dinucleotides, a process that is proposed to play a modulatory role in viral latency.

Cyclic adenosine monophosphate-responsive elements are involved in the transcriptional activation of the human IL-10 gene in monocytic cells.

IL-10 plays an important role in the regulation of immune responses. We and others have demonstrated recently that cyclic adenosine monophosphate (cAMP)-elevating substances up-regulate monocytic IL-10 expression in vitro and in vivo. Computer analysis of the IL-10 promoter/enhancer region localized four putative cAMP-responsive elements (CRE1- 4) with homology to the CRE consensus motif. In electrophoretic mobility shift assays CRE1 and CRE4 bound protein complexes consisting of transcription factors CREB-1 and ATF-1, while CRE3 bound only marginal amounts of CREB-1/ATF-1 in combination with unknown protein(s). CRE2 showed no protein binding activity. In vitro mutation of CRE1 and CRE4 reduced the level of cAMP-stimulated transactivation in reporter gene assays in comparison to the wild-type promoter by 20 % and 50 %, respectively, while mutation of CRE3 had no effect. The main action of CRE4 on cAMP-dependent stimulation is probably based on its adjacent localization to the TATA box and its sequence comprising a perfect half site. Experiments with double and triple mutants and with deleted promoter fragments indicated the participation of additional elements beside the CRE motifs in the cAMP-dependent stimulation. Our data suggest that intracellular cAMP may directly affect expression of the immunoregulatory cytokine IL-10 in monocytic cells via activation of the eukaryotic transcription factors CREB-1 and ATF-1 and their binding to CRE1 and CRE4 in the upstream enhancer of the IL-10 promoter.