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Biomedical subjects

H Jacobsen

Publications and source records attributed to H Jacobsen.

At least 55 records · Page 3Linked to original sources
PubMed1996

Reduced sensitivity to saquinavir: an update on genotyping from phase I/II trials.

A genotypic analysis of the HIV-1 proteinase was performed on clinical specimen obtained from patients after different periods of Saquinavir (SQV) treatment. Proteinase genes of integrated proviral DNA from PBMC were isolated by PCR, cloned and individual sequences were obtained. Genotypic resistance was defined by the Gly48-->Val and Leu90-->Met exchanges. Frequencies and kinetics of resistance development will be reported for phase I/II trials V13330. V13329, O13328 and ACTG229 in patients on monotherapy or combination therapy with RT inhibitors. Data from V13330 have been analysed in more detail for correspondence of genotypic and phenotypic resistance and any correlation between resistance and changes in plasma viral RNA load. Furthermore, we will discuss the data from our extensive proteinase gene sequence collection with respect to mutational changes which would be indicative of resistance to other inhibitors of HIV-1 proteinase.

Drug Resistance, Microbial↗
PubMed1996

Purification and in vitro-phospholabeling of secretory envelope proteins E1 and E2 of hepatitis C virus expressed in insect cells.

The putative envelope glycoproteins of hepatitis C virus (HCV), E1 and E2, were expressed as recombinant, secretory proteins in Sf9 insect cells through infection with recombinant baculoviruses. The influenza virus hemagglutinin signal sequence (HASS) was inserted upstream of the HCV-cDNAs in order to effect secretion. Furthermore, a hexa-histidine tag for purification on a Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) column and a protein kinase A (PKA) recognition sequence for in vitro-phospholabeling were fused upstream of the HCV-cDNA. E1- and E2 proteins lacking their carboxy-terminal, hydrophobic sequence were produced by baculovirus-infected insect cells in bioreactors of 23 1. The medium was concentrated and proteins were purified under native conditions on Ni(2+)-NTA columns. Purified proteins could be phospholabeled in vitro using the catalytic subunit of protein kinase. A isolated from bovine heart and gamma-[32P]ATP. Labeled E1 and E2 proteins expressed in insect cells could be immunoprecipitated with sera from HCV-infected patients. Co-expression of these E1 and E2 proteins led to the formation of E1-E2 complexes within the insect cell and to secretion of these complexes into the medium.

Adenosine Triphosphate↗
PubMed1996

In vivo resistance to a human immunodeficiency virus type 1 proteinase inhibitor: mutations, kinetics, and frequencies.

Resistance to saquinavir (Ro 31-8959), an inhibitor of human immunodeficiency virus type I proteinase, was studied in peripheral blood mononuclear cell-derived proviral DNA from patients undergoing prolonged treatment. A Leu90-->Met exchange was the predominant resistance mutation in vivo; Gly48-->Val or doubly mutant virus was rarely observed. After 8-12 months of treatment with saquinavir alone (600 mg, 3 times/day) or in combination with zidovudine (200 mg, 3 times/day), approximately 45% of all patients carried provirus with mutant proteinase; the incidence was lower (22%) in patients treated with a combination of saquinavir, zidovudine, and dideoxycytidine. There was a good relationship between genotypic analysis of saquinavir resistance and data from virus assays, confirming that Leu90-->Met and Gly48-->Val are the essential exchanges in the proteinase that determine loss of sensitivity to this inhibitor. Absence of genotypic resistance correlated with a sustained decrease in plasma viral RNA. There was a positive correlation between a Met90 mutation and some residues at natural polymorphic sites (positions 10, 36, 63, and 71).

Antiviral Agents↗
PubMed1995

An in vitro assay for hepatitis C virus NS3 serine proteinase.

Hepatitis C virus (HCV) encodes a polyprotein of which the majority of nonstructural proteins are matured by the viral serine proteinase located in the N terminus of NS3. Intracellular studies using recombinant vaccinia virus have shown that both NS3 and its cofactor NS4A are required to enhance processing at the NS3-dependent cleavage sites. We developed an in vitro (cell-free) assay in which the HCV serine proteinase was shown to be enzymatically active, by mixing lysates of cells expressing either the serine proteinase or a nonstructural protein substrate. NS3 cleaved in a highly reproducible manner at the NS5A/5B site in the presence of NS4A, whereas NS3 alone was enzymatically inactive. NS4A could be provided either linked to NS3 or as part of the substrate. In contrast, irrespective of the presence or absence of NS4A, no NS3-mediated processing was observed at the NS3/4A, NS4A/4B, and NS4B/5A sites in this assay. In vitro cleavage at the NS5A/5B site occurred rapidly, within 1 min at temperatures ranging from 0 to 20 degrees, but was incomplete and required detergent-solubilized lysates. General serine proteinase inhibitors did not decrease processing activity. The in vitro model described in this study is a new tool: (1) to study the structure and the function of HCV serine proteinase and NS5A/5B cleavage site, and (2) to test NS3 serine proteinase inhibitors.

Binding Sites↗
PubMed1995

Characterization of human immunodeficiency virus type 1 mutants with decreased sensitivity to proteinase inhibitor Ro 31-8959.

A human immunodeficiency virus type 1 (HIV-1) variant with highly reduced susceptibility to Ro 31-8959, an inhibitor of the viral proteinase, has been selected by repeated passage of wild-type virus in CEM cells in the presence of increasing concentrations of the inhibitor. Peptide sequences of the proteinase of selected virus were obtained from proviral DNA. Sequence comparison to wild-type (wt) proteinase demonstrated two amino acid substitutions in the resistant virus, a Gly to Val exchange at position 48 and a Leu to Met exchange at position 90. Furthermore, sequences of intermediate passage virus suggest contributions from positions 12, 36, 57, and 63 in early steps of resistance development. The selected virus showed a ca. 40-fold increase in 50% inhibitory concentration of Ro 31-8959. Growth kinetics of resistant virus were comparable to wild-type virus and the resistant genotype proved to be stable in the absence of inhibitor. Directed mutagenesis of the HIV-1 HXB2 proteinase at positions 48 and 90 suggested that each mutation alone led to a moderate decrease in sensitivity of the recombinant virus to proteinase inhibitor. However, a recombinant virus carrying both mutations in the proteinase gene showed a significant reduction in its sensitivity to Ro 31-8959 thus proving the importance of these exchanges for the resistance phenotype.

Amino Acid Sequence↗
PubMed1995

Substrate determinants for cleavage in cis and in trans by the hepatitis C virus NS3 proteinase.

Processing of the hepatitis C virus polyprotein is accomplished by a series of cotranslational and posttranslational cleavages mediated by host cell signalases and two virally encoded proteinases. Of these the NS3 proteinase is essential for processing at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions. Processing between NS3 and NS4A occurs in cis, implying an intramolecular reaction mechanism, whereas cleavage at the other sites can also be mediated in trans. Sequence analysis of the amino termini of mature cleavage products and comparisons of amino acid residues around the scissile bonds of various hepatitis C virus isolates identified amino acid residues which might contribute to substrate specificity and processing efficiency: an acidic amino acid at the P6 position, a Thr or Cys at the P1 position, and a Ser or Ala at the P1' position. To study the importance of these residues for NS3-mediated cleavage we have undertaken a mutational analysis using an NS3'-5B polyprotein expressed by recombinant vaccinia viruses in mammalian cells. For all NS3-dependent cleavage sites P1 substitutions had the most drastic effects on cleavage efficiency, showing that amino acid residues at this position are the most critical substrate determinants. Since less drastic effects were found for substitutions at the P1' position, these residues appear to be less important for proper cleavage. For all cleavage sites the P6 acidic residue was dispensable, suggesting that it is not essential for substrate recognition and subsequent cleavage. Analysis of a series of mutations at the NS3/4A site revealed great flexibility for substitutions compared with more stringent requirements at the trans cleavage sites. On the basis of these results we propose a model in which processing in cis is determined primarily by polyprotein folding, whereas cleavage in trans is governed not only by the structure of the polyprotein but also by specific interactions between the proteinase and the polyprotein substrate at or around the scissile bond.

Amino Acid Sequence↗
PubMed1994

Interruption of rotavirus spread through chemical disinfection.

INTRODUCTION: Rotaviruses, which are among the most important infectious causes of acute diarrhea, frequently cause outbreaks in hospitals, daycare centers, schools, and nursing homes. These viruses can remain viable on inanimate surfaces for many days and infectious rotavirus particles have been recovered from hands and a variety of surfaces and objects. Casual contact can lead to the transfer of these viruses from contaminated to clean surfaces. Therefore, animate and inanimate surfaces may play a complementary role in the spread of these viruses. OBJECTIVE: In this study, we compared the capacity of a disinfectant spray (0.1% o-phenylphenol and 79% ethanol), a domestic bleach (6% sodium hypochlorite diluted to give 800 ppm free chlorine), a quarternary ammonium (quat)-based product (7.05% quat diluted 1:128 in tap water), and a phenol-based product (14.7% phenol diluted 1:256 in tap water) to interrupt the transfer of a human rotavirus (DS-1) from stainless steel disks to fingerpads of volunteers with a 10-second contact at a pressure of 1 kg/cm2. DESIGN: Each disk received a 10 microL inoculum containing 1.0 x 10(4) to 7.0 x 10(4) plaque-forming units (PFU) of the virus suspended in 10% feces. The inoculum was dried for 1 hour and overlaid with 20 microL of either tap water or the test product. RESULTS: A 10-minute exposure to tap water reduced the virus titer by 52.3% +/- 11.7%. The disinfectant spray was able to reduce virus infectivity by > 99.99% after a contact of 3 to 10 minutes. The loss in virus infectivity after a 10-minute treatment with the quat was almost the same (54.7% +/- 17.8%) as seen with tap water. The activities of the bleach and the phenolic were very similar with losses in PFU of 97.9% +/- 0.4% and 95% +/- 5.36%, respectively. No detectable virus was transferred to fingerpads from disks treated with disinfectant spray, the bleach, and the phenolic. Contact of the fingerpads with tap water- or quat-treated disks resulted in the transfer of 5.6% +/- 1.1% and 7.6% +/- 2.5% of the remaining infectious virus, respectively. CONCLUSION: These findings emphasize the care needed in the selection of environmental surface disinfectants in preventing the spread of rotaviral infections.

Adult↗
PubMed1994

Performance of third-generation confirmatory tests for detection of antibody to hepatitis C virus.

We investigated three immunoblot assays (RIBA 3.0 from Chiron, Matrix from Abbott Laboratories, and LiaTek III from Organon Teknika) for the detection of antibody to hepatitis C virus. RIBA 3.0 and Matrix require reactivity to two antigens and LiaTek III requires reactivity to one for a sample to be positive. We tested 80 samples that were positive in repeat enzyme immunoassays by supplemental tests. The results showed that 55, 46, and 28% were reactive by LiaTek III, RIBA 3.0, and Matrix, respectively; 54, 33, and 13% of the samples were indeterminate by Matrix, RIBA 3.0, and LiaTek III, respectively. There were 32, 21, and 16% nonreactive samples by LiaTek III, RIBA 3.0, and Matrix, respectively. Of the samples positive by RIBA 3.0, only 50 and 76% were reactive by Matrix and LiaTek III, respectively. A large number of samples that were indeterminate by RIBA 3.0 were positive by LiaTek III (52%). The core antigen was the most reactive antigen in all three tests (48 to 57%). The NS4 antigen in Matrix (20%) and LiaTek III (16%) was poorly reactive, although it performed better in RIBA 3.0 (45%). The NS5 and E2/NS1 antigens made minor contributions to reactivity. The combinations of the core, NS3, and NS4 antigens produced 77% of the RIBA 3.0 and 100% of the Matrix reactive samples. The results showed a poor correlation among the three tests.

Antigens, Viral↗
PubMed1994

Kinetic and structural analyses of hepatitis C virus polyprotein processing.

Recombinant vaccinia viruses were used to study the processing of hepatitis C virus (HCV) nonstructural polyprotein precursor. HCV-specific proteins and cleavage products were identified by size and by immunoprecipitation with region-specific antisera. A polyprotein beginning with 20 amino acids derived from the carboxy terminus of NS2 and ending with the NS5B stop codon (amino acids 1007 to 3011) was cleaved at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B sites, whereas a polyprotein in which the putative active site serine residue was replaced by an alanine remained unprocessed, demonstrating that the NS3-encoded serine-type proteinase is essential for cleavage at these sites. Processing of the NS3'-5B polyprotein was complex and occurred rapidly. Discrete polypeptide species corresponding to various processing intermediates were detected. With the exception of NS4AB-5A/NS5A, no clear precursor-product relationships were detected. Using double infection of cells with vaccinia virus recombinants expressing either a proteolytically inactive NS3'-5B polyprotein or an active NS3 proteinase, we found that cleavage at the NS4A/4B, NS4B/5A, and NS5A/5B sites could be mediated in trans. Absence of trans cleavage at the NS3/4A junction together with the finding that processing at this site was insensitive to dilution of the enzyme suggested that cleavage at this site is an intramolecular reaction. The trans-cleavage assay was also used to show that (i) the first 211 amino acids of NS3 were sufficient for processing at all trans sites and (ii) small deletions from the amino terminus of NS3 selectively affected cleavage at the NS4B/5A site, whereas more extensive deletions also decreased processing efficiencies at the other sites. Using a series of amino-terminally truncated substrate polyproteins in the trans-cleavage assay, we found that NS4A is essential for cleavage at the NS4B/5A site and that processing at this site could be restored by NS4A provided in cis (i.e., together with the substrate) or in trans (i.e., together with the proteinase). These results suggest that in addition to the NS3 proteinase, NS4A sequences play an important role in HCV polyprotein processing.

Binding Sites↗
PubMed1994

Sjögren's syndrome in inflammatory rheumatic diseases: analysis of the leukocyte protein calprotectin in plasma and saliva.

In a hospital population of 154 patients with a wide range of inflammatory rheumatic diseases, patients with sicca symptoms were subjected to objective ocular and oral tests to establish cases with Sjögren's syndrome (SS). The plasma level of the leukocyte protein calprotectin has been shown to be a good indicator of disease activity and inflammation in various rheumatic diseases. In the present study, calprotectin levels in plasma and whole saliva were analysed and evaluated as potential markers of SS and salivary gland disease activity. Plasma calprotectin levels did not differ significantly between patients with SS and patients with no sicca symptoms. However, salivary calprotectin levels correlated significantly with the plasma calprotectin levels and with several ocular variables, weakly with salivary flow and serum rheumatoid factor, but not with focal sialadenitis. In conclusion, this study shows that salivary calprotectin levels seem to be associated with several variables of SS glandular pathology, indicating the need for further and more comprehensive studies on calprotectin in various oral fluids and in lacrimal fluid in relation to SS glandular disease activity.

Blood↗
PubMed1994

Identification of an amino acid substitution involved in the reduction of sensitivity of HIV-1 to an inhibitor of viral proteinase.

Clones derived from HIV variants previously characterized as resistant to Ro31-8959, an inhibitor of viral proteinase (PR), were sequenced. Substitution of glycine by valine at position 48 of the PR protein was found. None of the 20 clones derived from wild type HTLV-IIIB contain this mutation. Since such a position is located in a conserved region of PR, it is possible that the substitution can affect the interaction of the enzyme with the inhibitor.

Base Sequence↗
PubMed1993

Chemical disinfection to interrupt transfer of rhinovirus type 14 from environmental surfaces to hands.

Rhinoviruses can survive on environmental surfaces for several hours under ambient conditions. Hands can readily become contaminated after contact with such surfaces, and self-inoculation may lead to infection. Whereas hand washing is crucial in preventing the spread of rhinovirus colds, proper disinfection of environmental surfaces may further reduce rhinovirus transmission. In this study, the capacities of Lysol Disinfectant Spray (0.1% o-phenylphenol and 79% ethanol), a domestic bleach (6% sodium hypochlorite diluted to give 800 ppm of free chlorine), a quaternary ammonium-based product (7.05% quaternary ammonium diluted 1:128 in tap water), and a phenol-based product (14.7% phenol diluted 1:256 in tap water) were compared in interrupting the transfer of rhinovirus type 14 from stainless steel disks to fingerpads of human volunteers upon a 10-s contact at a pressure of 1 kg/cm2. Ten microliters of the virus, suspended in bovine mucin (5 mg/ml), was placed on each disk, and the inoculum was dried under ambient conditions; the input number on each disk ranged from 0.5 x 10(5) to 2.1 x 10(6) PFU. The dried virus was exposed to 20 microliters of the test disinfectant. The Lysol spray was able to reduce virus infectivity by > 99.99% after a contact of either 1 or 10 min, and no detectable virus was transferred to fingerpads from Lysol-treated disks. The bleach (800 ppm of free chlorine) reduced the virus titer by 99.7% after a contact time of 10 min, and again no virus was transferred from the disks treated with it.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions.

We have studied processing of the nonstructural (NS) polyprotein of the hepatitis C virus. A series of cDNAs corresponding to predicted NS2/3/4 or NS3/4 regions were constructed, and processing of the polyproteins was studied in an in vitro transcription-translation system. We report that a catalytically active serine-type proteinase is encoded by the NS3 region. Substitution of the serine residue of the putative catalytic triad (H, D, and S) by alanine blocked cleavage at the NS3/4 junction, while processing between NS2 and NS3 was not affected. Thus, cleavage at the NS2/3 junction is mediated either by cellular enzymes or by an NS-2 inherent proteinase activity. Deletion analysis of an NS3/4 cDNA construct mapped the amino terminus of the enzymatically active proteinase between amino acids 1049 and 1065 of the polyprotein. As internal deletions of variable segments of the presumed helicase domain prevented processing at the NS314 junction, a continuous NS3 region appears to be required for processing at this site. To analyze hepatitis C virus polyprotein cleavage in vivo, recombinant vaccinia viruses expressing NS2/3/4 or NS3/4/5 proteins were generated. In agreement with the in vitro data, cleavage between NS2 and NS3 was independent of a catalytically active NS3 proteinase, whereas substitution of the active-site serine residue by the amino acid alanine completely blocked processing at the NS3/4 and NS4/5 junctions. These results demonstrate that NS3 encodes the viral proteinase essential for generating the amino termini of NS4 and NS5.

Amino Acid Sequence↗
PubMed1992

[Peripheral facial paresis as a symptom of Borrelia burgdorferi infection].

Infection with the tick-borne Borrelia burgdorferi can lead to a variety of neurologic symptoms, the most frequent being a radicular pain syndrome due to meningitis. General symptoms such as asthenia or headache are also frequent, however, and serious neurologic complications such as dementia or spastic paresis may occur. At an early stage, Borrelia infections can be easily treated with antibiotics, which makes it important to recognize the symptoms and make the correct diagnosis. A common feature of borreliosis is facial palsy, and in the article is described the case of a 14 year-old boy with borreliosis and bilateral facial palsy. The frequency of facial palsy from borreliosis is probably high. The authors discuss the indications for performing lumber puncture in patients with apparent idiopathic facial palsy (Bell's palsy).

Adolescent↗
PubMed1992

Progression of early steps of human immunodeficiency virus type 1 replication in the presence of an inhibitor of viral protease.

We have evaluated a possible role for human immunodeficiency virus type 1 protease during early steps of replication. For these studies, a specific inhibitor of human immunodeficiency virus protease, Ro31-8959, was used. Synthesis of viral cDNA, its integration into cellular DNA, and its transcription were determined during a one-step, acute infection of MT-4 cells. No consistent difference in any of these parameters was noted between control-infected cultures and those treated with protease inhibitor. However, no infectious progeny virus was produced in treated cultures, and thus spread of infection was severely restricted. Our results do not support an essential activity of viral protease in early steps of replication but are in line with its established role in gag and gag-pol processing and in maturation to infectious progeny virus.

Antiviral Agents↗
PubMed1990

Different effects of IFN gamma and IFN alpha/beta on "immediate early" gene expression of HSV-1.

The effects of interferon gamma (IFN gamma) on early steps of herpes simplex virus (Type 1; HSV-1) replication in primary cultures of splenic mouse macrophages were analyzed and compared to IFN alpha/beta. Pretreatment of macrophages with recombinant murine IFN gamma led to a dose-dependent reduction in the yield of progeny virus. Inhibition of protein synthesis was observed for HSV-1 alpha, beta and gamma-proteins. Expression of the "immediate early" (IE) gene IE3 (ICP4) was investigated in detail. Steady-state level of the RNA and transcriptional activity of the gene in IFN gamma-treated cells were comparable to control-infected cells except for a delay in their kinetics. This is in contrast to IFN alpha/beta, which leads to a stable decrease in IE3 transcripts. Since IFN gamma causes a stable decrease in the IE3 gene product ICP4, our data suggest a translational inhibition of HSV-1 IE gene expression in IFN gamma-treated macrophages. Thus, IFN gamma and IFN alpha/beta inhibit HSV-1 replication by different mechanisms which may lead to a synergistic enhancement of inhibition after combined treatment.

Animals↗
PubMed1990

The urethral plug: a new treatment modality for genuine urinary stress incontinence in women.

A new modality, the urethral plug, was used to treat 22 women with genuine urinary stress incontinence. The plug is made of thermoplastic elastomer (Kraton G), and consists of a meatal plate, a soft stalk and 1 or 2 spheres along the stalk. The spheres were located according to the result of the urethral pressure profile. The midpoint of the proximal sphere was placed at the bladder neck and the distal sphere was placed just above the maximum urethral pressure point. At voiding the plug was removed and afterwards a new plug was inserted. The plug with 2 spheres was tested in week 1 (period 1) and the plug with only the distal sphere was tested in week 2 (period 2). A total of 22 patients completed period 1. Eight patients did not complete period 2, mostly due to either unchanged incontinence during period 1 or a repeated loss of the plug with 1 sphere. In periods 1 and 2, 73 and 79% of the patients were subjectively and objectively continent or improved. A total of 14 patients completed both periods. Eight patients preferred the plug with 2 spheres, 1 preferred the other plug and 5 had no preference. The side effects were few. This preliminary study shows that the urethral plug seems to be a promising alternative treatment for female genuine urinary stress incontinence.

Equipment Design↗