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G Sutter

Publications and source records attributed to G Sutter.

At least 37 records · Page 2Linked to original sources
PubMed1998

Serp2, an inhibitor of the interleukin-1beta-converting enzyme, is critical in the pathobiology of myxoma virus.

Recently, myxoma virus was shown to encode an additional member of the serpin superfamily. The viral gene, called serp2, was cloned, and the Serp2 protein was shown to specifically bind to interleukin-1beta (IL-1beta)-converting enzyme (ICE), thus inhibiting the cleavage of pro-IL-1beta by the protease (F. Petit, S. Bertagnoli, J. Gelfi, F. Fassy, C. Boucraut-Baralon, and A. Milon, J. Virol. 70:5860-5866, 1996). Here, we address the role of Serp2 in the development of myxomatosis, a lethal infectious disease of the European rabbit. A Serp2 mutant myxoma virus was constructed by disruption of the single-copy serp2 gene and insertion of the Escherichia coli gpt gene serving as the selectable marker. A revertant virus was obtained by replacing the E. coli gpt gene by the intact serp2 open reading frame. The Serp2(-) mutant virus replicated with wild-type kinetics both in rabbit fibroblasts and a rabbit CD4(+) T-cell line (RL5). Moderate reduction of cell surface levels of major histocompatibility complex I was observed after infection with wild-type or Serp2(-) mutant myxoma virus, and both produced white pocks on the chorioallantoic membrane of the chick embryo. After the infection of European rabbits, the Serp2(-) mutant virus proved to be highly attenuated compared to wild-type myxoma virus, as demonstrated by the clinical course of myxomatosis and the survival rates of infected animals. Pathohistological examinations revealed that infection with wild-type myxoma virus resulted in a blockade of the inflammatory response at the vascular level. In contrast, rapid inflammatory reactions occurred upon infection with the Serp2(-) mutant virus. Furthermore, lymphocytes in lymph nodes derived from animals inoculated with Serp2 mutant virus were shown to rapidly undergo apoptosis. We postulate that the virulence of myxoma virus in the European rabbit can be partially attributed to an impairment of host inflammatory processes and to the prevention of apoptosis in lymphocytes. The weakening of host defense is directly linked to serp2 gene function and is likely to involve the inhibition of IL-1beta-converting-enzyme-dependent pathways.

Animals↗
PubMed1997

Rescue of measles virus using a replication-deficient vaccinia-T7 vector.

A system which allows the reconstitution of measles virus (MV) from cloned cDNA is described. The severely host cell restricted vaccinia vector MVA-T7 expressing bacteriophage T7 RNA polymerase was used to generate full-length antigenomic MV RNA and simultaneously the mRNAs encoding the viral N, P and L proteins in order to produce replicationally and transcriptionally active nucleocapsids. The functionality of the N, P and L proteins was demonstrated first by their ability to rescue MV specific subgenomic RNAs. Assembly and budding of reconstituted MV was shown by syncytia formation and subsequently by virus isolation. The inability of MVA-T7 to produce progeny virus in most mammalian cells circumvents the necessity to separate the reconstituted MV from the MVA-T7 helper virus. Since all components are expressed transiently, this system is especially suitable for studying the functions of N, P and L. Furthermore, it is useful for investigating later steps in the MV life cycle.

Animals↗
PubMed1997

Recognition of prominent viral epitopes induced by immunization with human immunodeficiency virus type 1 regulatory genes.

Mice immunized with the regulatory genes nef, rev, and tat from human immunodeficiency virus type 1 developed both humoral and cellular immune responses to the gene products Nef, Rev, and Tat. This study demonstrates that it is feasible to induce immune reactions to all of these regulatory gene products. Humoral responses were seen after DNA boosts, while potent T-cell proliferative responses were noted already after a single immunization. A Th1-directed immune response was demonstrated early after immunization. A 3- to 75-fold-stronger T-cell response was seen in animals receiving DNA epidermally compared to that in animals receiving intramuscular injections. Nef, Rev, and Tat putative B- and T-cell epitopes were clearly mapped by using peptides derived from the regulatory proteins and were similar to those which are detected in human immunodeficiency virus infection. Although immunization by the Nef, Rev, and Tat proteins raised high immunoglobulin G titers in serum, the epitope spreading appeared broader after DNA immunization. The combination of all of these regulatory genes together with two genes for structural proteins, the envelope and gag genes, demonstrated that a combined approach is feasible in that reactivities to all antigens persisted or were even augmented. No interference between plasmids was noted.

AIDS Vaccines↗
PubMed1996

Host range restricted, non-replicating vaccinia virus vectors as vaccine candidates.

Three model systems were used to demonstrate the immunogenicity of highly attenuated and replication-defective recombinant MVA. (1) Intramuscular inoculation of MVA-IN-Fha/np induced humoral and cell-mediated immune responses in mice and protectively immunized them against a lethal respiratory challenge with influenza virus. Intranasal vaccination was also protective, although higher doses were needed. (2) In rhesus macaques, an immunization scheme involving intramuscular injections of MVA-SIVenv/gag/pol greatly reduced the severity of disease caused by an SIV challenge. (3) In a murine cancer model, immunization with MVA-beta gal prevented the establishment of tumor metastases and even prolonged life in animals with established tumors. These results, together with previous data on the safety of MVA in humans, suggest the potential usefulness of recombinant MVA for prophylactic vaccination and therapeutic treatment of infectious diseases and cancer.

Animals↗
PubMed1996

Patterns of viral replication correlate with outcome in simian immunodeficiency virus (SIV)-infected macaques: effect of prior immunization with a trivalent SIV vaccine in modified vaccinia virus Ankara.

The dynamics of plasma viremia were explored in a group of 12 simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) that had received prior immunization with either nonrecombinant or trivalent (gag-pol, env) SIV-recombinant vaccinia viruses. Three distinct patterns of viral replication observed during and following primary viremia accounted for significant differences in survival times. High-level primary plasma viremia with subsequently increasing viremia was associated with rapid progression to AIDS (n = 2). A high-level primary plasma virus load with a transient decline and subsequent progressive increase in viremia in the post-acute phase of infection was associated with progression to AIDS within a year (n = 6). Low levels of primary plasma viremia followed by sustained restriction of virus replication were associated with maintenance of normal lymphocyte subsets and intact lymphoid architecture (n = 4), reminiscent of the profile observed in human immunodeficiency virus type 1-infected long-term nonprogressors. Three of four macaques that showed this pattern had been immunized with an SIV recombinant derived from the attenuated vaccinia virus, modified vaccinia virus Ankara. These data link the dynamics and extent of virus replication to disease course and suggest that sustained suppression of virus promotes long-term, asymptomatic survival of SIV-infected macaques. These findings also suggest that vaccine modulation of host immunity may have profound beneficial effects on the subsequent disease course, even if sterilizing immunity is not achieved.

Animals↗
PubMed1995

Non-replicating vaccinia vector efficiently expresses bacteriophage T7 RNA polymerase.

Modified vaccinia virus Ankara (MVA), a host range restricted and highly attenuated vaccinia virus strain, is unable to multiply in human and most other mammalian cell lines. Since viral gene expression is unimpaired in non-permissive cells recombinant MVA viruses are efficient as well as exceptionally safe expression vectors. We constructed a recombinant MVA that expresses the bacteriophage T7 RNA polymerase and tested its usefulness for transient expression of recombinant genes under the control of a T7 promoter. Using the chloramphenicol acetyltransferase (CAT) gene as a reporter gene, infection with MVA-T7pol allowed efficient synthesis of recombinant enzyme in mammalian cells. Despite the severe host restriction of MVA, enzyme activities induced by infection with MVA-T7pol were similar to those determined after infection with a replication-competent vaccinia-T7pol recombinant virus. Thus, MVA-T7pol may be used as a novel vaccinia vector to achieve T7 RNA polymerase-specific recombinant gene expression in the absence of productive vaccinia virus replication.

Animals↗
PubMed1995

In situ amplification of measles virus RNA by the self-sustained sequence replication reaction.

BACKGROUND: The self-sustained sequence replication (3SR) reaction is an isothermal method for nucleic acid amplification that has several features that make it an attractive alternative to PCR. We have studied the feasibility of the in situ 3SR reaction in cells using a measles virus-infected cell line as a model. EXPERIMENTAL DESIGN: The study was carried out in four steps. First, using RNA extracted from a measles-infected Vero Green monkey kidney cell line, conditions for the in vitro amplification of a segment of the nucleocapsid portion of the RNA viral genome were optimized for 420- and 119-bp 3SR products, and the results were compared. Second, 3SR was performed on intact infected cells in suspension, and the amount of RNA product was compared with infected cells without 3SR. Then, the 3SR reaction was conducted on cytospin preparation slides, followed by in situ hybridization for detection of the amplification product. Finally, 3SR was carried out on sections of formalin-fixed, paraffin-fixed, paraffin-embedded cells, and the degree of amplification as detected by ISH was quantified and compared between infected cells with and without 3SR reaction. RESULTS: Specific amplification of measles was observed in each of these types of preparations with an 8.5-fold rate of amplification in paraffin sections of formalin-fixed cells (a mean of 272.5 +/- 65.3 grains/cell after 3SR amplification in comparison to 31.97 +/- 4.2 grains/cell without amplification). CONCLUSIONS: A significant amount of amplification of RNA is possible with in situ 3SR (IS-3SR) and, in combination with ISH, offers several advantages compared with in situ PCR (IS-PCR), such as ease of use, lack of conditions that lead to cell damage, and a specificity for RNA amplification. This is the first report of specific amplification of RNA within cells using the IS-3SR procedure, a technique that has a wide range of potential applications in pathology and molecular biology.

Animals↗
PubMed1994

A cellular factor is required for transcription of vaccinia viral intermediate-stage genes.

The cytoplasmic location of vaccinia virus replication and evidence that the multisubunit DNA-dependent RNA polymerase, early and late stage transcription factors, capping and methylating enzymes, and poly(A) polymerase are virus encoded raised the possibility that all of the proteins needed for viral mRNA synthesis are of viral origin. Previous studies showed that four components from infected cells, the viral RNA polymerase and capping enzyme and two factors called vaccinia virus intermediate transcription factors (VITFs) 1 and 2, can reconstitute transcription of vaccinia virus intermediate-stage genes in vitro. Here, we demonstrate that VITF-2 can be isolated from the nuclei of uninfected HeLa cells as well as from the cytoplasm of infected cells. The proteins with VITF-2 activity from uninfected and infected cells cochromatographed and cosedimented, suggesting that they are identical. VITF-2 activity was found in extracts of other uninfected human and monkey cells but not in nonpermissive Trichoplusia ni insect cells or in conditionally permissive rabbit kidney 13 cells. VITF-2 activity was present, however, in a permissive line of rabbit kidney 13 cells that had been stably transfected with the vaccinia virus K1L host range gene. We suggest that the VITF-2 level acts as a gauge of the permissive state of the cell and thereby regulates the length of the early prereplicative phase of the infection.

Cell Cycle Proteins↗
PubMed1994

A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus.

The immunogenicity of a recombinant virus derived from modified vaccinia virus Ankara (MVA), a host range-restricted, highly attenuated and safety-tested strain, was investigated. Plasmid transfer vectors that provide strong synthetic early/late promoters for the simultaneous expression of two genes as well as a transient or stable selectable marker and flanking sequences for homologous recombination with the MVA genome were constructed. A recombinant MVA containing influenza virus haemagglutinin and nucleoprotein genes was isolated in avian cells and shown to express both proteins efficiently upon infection of human or mouse cells in which abortive replication occurs. Mice, inoculated by various routes with recombinant MVA, produced antibody and cytotoxic T-lymphocyte responses to influenza virus proteins and were protected against a lethal influenza virus challenge as effectively as mice immunized with a recombinant derived from the replication-competent WR strain of vaccinia virus.

Animals↗
PubMed1994

Stable expression of the vaccinia virus K1L gene in rabbit cells complements the host range defect of a vaccinia virus mutant.

Modified vaccinia virus Ankara (MVA), having acquired genomic deletions during passage in chicken embryo fibroblasts, is highly attenuated and unable to productively infect most mammalian cell lines. Multiplication in rabbit kidney-derived RK13 cells, but not other nonpermissive cells, can be restored by insertion of the vaccinia virus K1L gene into the MVA genome. During nonproductive infection of RK13 cells by MVA, transcription of representative viral early genes was revealed by Northern (RNA) blotting, whereas synthesis of an intermediate mRNA and replication of viral DNA could not be detected. Despite the persistence of viral early mRNA for at least several hours, synthesis of virus-induced polypeptides occurred only during the first hour and was followed by abrupt inhibition of all protein synthesis. Transfection of RK13 cells with a eukaryotic expression plasmid that contained the K1L gene allowed MVA infection to proceed to late stages of viral protein synthesis. Moreover, RK13 cell lines that stably expressed the K1L gene were permissive for MVA as well as a K1E deletion mutant of the WR strain of vaccinia virus. This is the first description of the complementation of a poxvirus mutant by cells that stably express a viral gene.

Animals↗
PubMed1992

Nonreplicating vaccinia vector efficiently expresses recombinant genes.

Modified vaccinia Ankara (MVA), a highly attenuated vaccinia virus strain that has been safety tested in humans, was evaluated for use as an expression vector. MVA has multiple genomic deletions and is severely host cell restricted: it grows well in avian cells but is unable to multiply in human and most other mammalian cells tested. Nevertheless, we found that replication of viral DNA appeared normal and that both early and late viral proteins were synthesized in human cells. Proteolytic processing of viral structural proteins was inhibited, however, and only immature virus particles were detected by electron microscopy. We constructed an insertion plasmid with the Escherichia coli lacZ gene under the control of the vaccinia virus late promoter P11, flanked by sequences of MVA DNA, to allow homologous recombination at the site of a naturally occurring 3500-base-pair deletion within the MVA genome. MVA recombinants were isolated and propagated in permissive avian cells and shown to express the enzyme beta-galactosidase upon infection of nonpermissive human cells. The amount of enzyme made was similar to that produced by a recombinant of vaccinia virus strain Western Reserve, which also had the lacZ gene under control of the P11 promoter, but multiplied to high titers. Since recombinant gene expression is unimpaired in nonpermissive human cells, MVA may serve as a highly efficient and exceptionally safe vector.

Animals↗
PubMed1991

Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence.

Different passages of the vaccinia virus strain Ankara (CVA wild-type) during attenuation to MVA (modified vaccinia virus Ankara) have been analysed to detect alterations in the genome. Physical maps for the restriction enzymes HindIII and XhoI have been established. Six major deletions relative to the wild-type strain CVA could be localized. They reduce the size of the entire genome from 208 kb (CVA wild-type) to 177 kb for the MVA strain. Four deletions occurred during the first 382 passages and the resulting variant (CVA 382) displays an attenuated phenotype similar to that of the MVA strain. The deletions are located in both terminal fragments, affect two-thirds of the host range gene K1L and eliminate 3.5 kb of a highly conserved region in the HindIII A fragment. During the next 190 passages leading to MVA two additional deletions appeared. Again, one is located in the left terminal fragment, and the other includes the A-type inclusion body gene. Neither of the deletions appear to participate in further attenuation of the virus. Rescue of the partially deleted host range region with the corresponding wild-type DNA restored the ability of the attenuated strains MVA and CVA 382 to grow in some non-permissive tissue cultures. Nevertheless, the complete host range of the wild-type strain was not recovered. Also, plaque-forming behaviour and reduced virulence were not influenced. From the data presented it may be concluded that the partially deleted host range gene is not solely responsible for attenuation.

Animals↗
PubMed1991

Transferred triage to a level I trauma center in a mass catastrophe of patients; many of them with burns.

Three airplanes were involved in an airshow accident at the Ramstein military airbase on 28th of August 1988 causing immediate 45 dead and approximate 400 injured people. As a Level I Trauma facility we received 47 patients in different state of distress direct from airfield within an hour. The disaster plan was activated and sufficient personal and infrastructure could be mobilized.

Accidents, Aviation↗
PubMed1990

Formation of free radicals and nitric oxide derivative of hemoglobin in rats during shock syndrome.

Free radicals have been postulated to play an important role as mediators in the pathogenesis of shock syndrome and multiple-organ failure. We attempted to directly detect the increased formation of radicals by Electron Spin Resonance (ESR) in animal models of shock, namely the endotoxin (ETX) shock or the hemorrhagic shock of the rat. In freeze-clamped lung tissue, a small but significant increase of a free radical signal was detected after ETX application. In the blood of rats under ETX shock, a significant ESR signal with a triplet hyperfine structure was observed. The latter ESR signal evolved within several hours after the application of ETX and was localized in the red blood cells. This signal was assigned to a nitric oxide (NO) adduct of hemoglobin with the tentative structure [alpha 2+ NO)beta 3+)2. The amount of hemoglobin-NO formed, up to 0.8% of total hemoglobin, indicated that under ETX shock a considerable amount of NO was produced in the vascular system. This NO production was strongly inhibited by the arginine analog NG-monomethyl-arginine (NMMA). The ESR signal of Hb-NO was also observed after severe hemorrhagic shock. There are three questions, namely (i) the type of vascular cells and the regulation of the process forming such a large amount of NO during ETX shock, (ii) the pathophysiological implications of the formed NO, effects which have been described as cytotoxic mediator, endothelium-derived relaxing factor (EDRF) or inhibitor of platelet aggregation, and (iii) the possible use of Hb-NO for monitoring phases of shock syndrome.

Animals↗
PubMed1989

Serum mediated depression of chemiluminescence response of granulocytes in hemorrhagic shock.

CL of PMNL was decreased in the early stage of hemorrhagic shock in the rat if evaluated from whole blood. When standardized PMNL were incubated with plasma of different shock states the same serum--mediated depression was observed. In all stages killing activity of PMNL per se from arterial blood (after passage of pulmonary endothelium) was nearly unchanged if measured in standardized pool plasma. Furthermore in the prefinal stage of uncompensated shock highly activated PMNL were obtained. These results support the hypothesis that plasmatic--cellular interactions play an important role in depression of killing activity of PMNL already in early hemorrhagic shock. Actual concepts mention protein cleavage products, fluid phase C3b and prostaglandins as potential mediators of this serum--mediated suppression.

Animals↗
PubMed1988

Thromboxane receptor blockade in an animal model of ARDS.

Manipulation of arachidonic acid metabolism may be important in the prevention and treatment of the adult respiratory distress syndrome. This study evaluated a thromboxane receptor blocker, BM 13.177, in a sheep endotoxin model. Sheep with chronic lung lymph fistulas were pretreated with the blocker before being given endotoxin. The blocker attenuated the early increase in pulmonary artery pressure usually seen after endotoxin (blocker + endotoxin, 31.0 +/- 14.5 mm Hg; endotoxin alone, 42.1 +/- 9.6 mm Hg). There was no effect on lymph flows during the later (permeability) phase (at 6 hours: blocker + endotoxin, 574 +/- 287% baseline; endotoxin alone, 311 +/- 102% baseline). Administration of the blocker alone increased pulmonary artery pressure, arterial pressure, and central venous pressure and decreased heart rate and cardiac index. Thromboxane receptor blockade presumably has little effect on elements of arachidonic acid metabolism other than thromboxane. Thromboxane appears to be important in the early pulmonary hypertension of the sheep endotoxin model but is relatively unimportant in the development of increased pulmonary capillary permeability.

Animals↗
PubMedDate not supplied

An anti-HIV strategy combining chemotherapy and therapeutic vaccination.

Combination chemotherapy using potent anti-retroviral agents has led to significant advances in the clinical management of human immunodeficiency virus (HIV) disease. However, the emergence of multiple drug-resistant mutants, the high need for compliance to adhere to demanding drug-dosing schemes, and the remaining toxic side-effects of drugs make the perspective of life-long treatment unattractive and possibly unrealistic. Therefore, means must be sought to shorten the time span during which treatment is necessary. Such means could be to stimulate an efficient immune response during the period of low virus load and restored CD4 + cell levels, which might be capable of keeping the virus under long-lasting control after treatment is stopped. Here we tested this concept of combined chemotherapy/ therapeutic vaccination in a non-human primate model. Rhesus macaques chronically infected with the chimeric simian/human immunodeficiency virus (SHIV) containing the HIV type 1 (HIV-1) HXBc2 gene for reverse transcriptase (RT) in the genomic background of simian immunodeficiency virus (SIV)(mac239) (RT-SHIV) were treated with (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA), a potent anti-HIV drug. When virus load had decreased significantly, we immunized with SIV genes env, gag/pol, rev, tat, and nef inserted in two different expression vector systems. Four weeks after the second immunization, drug treatment was stopped. Animals were monitored to determine if virus load stayed low or if it increased again to the original levels and if CD4+ T-cell levels remained stable. Humoral and cellular immune responses were also measured. This combined chemotherapy/ therapeutic vaccination regimen induced a significant reduction in the steady-state level of viremia in one out of two chronically infected rhesus macaques. Chemotherapeutic treatment alone did not achieve reduction of viremia in two chronically infected animals. The nature of the immune responses assumed to have been induced by vaccination in one out of the two monkeys remains to be elucidated.

AIDS Vaccines↗