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Biomedical subjects

J E Hansen

Publications and source records attributed to J E Hansen.

At least 19 recordsLinked to original sources

An O-linked carbohydrate neutralization epitope of HIV-1 gp 120 is expressed by HIV-1 env gene recombinant vaccinia virus.

Previous studies have disagreed about the presence of O-linked carbohydrate epitopes on gp 120 of HIV, although antibodies against short-chain O-linked glycans neutralize HIV infection and block syncytium formation in vitro. To settle this question, we analysed the O-linked glycans of gp 120 by chemical methods using purified HIV-1 gp 120 from cells infected with recombinant vaccinia virus solely expressing gp 160 or gp 120. Alkaline borohydride degradation of recombinant gp 120 released monosaccharides and also slightly larger structures (di/trisaccharides) by a beta-elimination, confirming the presence of simple O-linked oligosaccharides. The functional activity as neutralisation epitopes of the O-linked oligosaccharides expressed on recombinant gp 120 was preserved, since fusion between uninfected CD4+ cells and cells infected with recombinant vaccinia was blocked by monoclonal antibodies to the O-linked oligosaccharides of gp 120. Although the mechanism for HIV induction of O-linked oligosaccharide neoantigens is unknown, these results indicate that the O-linked neutralization epitopes are inherent to the glycoprotein itself, and that the unusual appearance of simple O-linked oligosaccharides on gp 120 is independent of any interaction between the host cell and retroviral genes other than env.

Animals

Sensitive non-radioactive detection of HIV-1: use of nested primers for the amplification of HIV DNA.

This report describes the use of the polymerase chain reaction (PCR) for the non-radioactive detection of HIV-1 proviral genomic sequences in HIV-1 infected cells. We have developed a sensitive assay, using three different sets of nested primers and our results show that this method is superior to standard PCR for the detection of HIV-1 DNA. The assay described features the use of a simple and inexpensive sample preparation technique and a non-radioactive hybridization procedure for confirmation of results. To test the suitability of the assay for clinical purposes, we tested cell samples from 76 anti-HIV-1 positive patients. All were positive for at least one primer set: 88% were positive for all three sets of primers; 9% were positive for two sets of primers and 3% were positive for only one set of primers. It provides a useful approach to the study of HIV-1 infection in patient samples where genomic copies often are present at such low numbers that they are otherwise undetectable.

Base Sequence

Carbohydrate determinant NeuAc-Gal beta (1-4) of N-linked glycans modulates the antigenic activity of human immunodeficiency virus type 1 glycoprotein gp120.

In the present study we investigated to what extent the peripheral carbohydrate structure of N-linked glycans influences the antigenic properties of human immunodeficiency virus type 1 glycoprotein 120 (gp120). Recombinant gp120 was purified from GMK cells infected with a recombinant vaccinia virus expressing gp120. Purified gp120 was then coated onto 96-well ELISA microplates and subjected to sequential removal of peripheral monosaccharide units. Modified or unmodified gp120 was then incubated with monoclonal antibodies recognizing specific epitopes of gp120 and with a reporter lectin to determine the extent of carbohydrate elimination. Antibody and lectin binding was quantified in an enzyme-linked system. We found that the carbohydrate structure NeuAc-Gal beta (1-4) of N-linked glycans, defined both by lectin reactivity and by specific glycosidases, is involved in modulating the binding of antibody to a number of epitopes of peptide nature. The binding of antibody to one class of epitopes, situated in a region between amino acids 200 and 230, was strongly increased by removal of NeuAc-Gal beta (1-4), whereas the binding to epitopes in the V3 region was decreased and the binding to epitopes in the far N-terminal region was not altered by the treatment. These results suggested that peripheral structures of N-glycans are involved in modulating the overall conformation of gp120.

Antibodies, Monoclonal

The typing of Staphylococcus epidermidis by a lectin-binding assay.

A new typing method for Staphylococcus epidermidis was developed. Four biotinylated lectins--wheat germ agglutinin (WGA), soy bean agglutinin (SBA), lentil agglutinin (LCA) and Concanavalin A (ConA)--were added to immobilised whole cells of coagulase-negative staphylococci (CNS) in microtitration plates. The amount of bound lectin was measured by peroxidase-conjugated avidin followed by a peroxidase reaction. The method was compared to antibiotic-resistance analysis, phage typing, plasmid DNA profiles and slime production. A total of 113 isolates of CNS from 21 patients was investigated and 71 strains of CNS, including 64 strains of S. epidermidis, were detected if all typing methods were taken into consideration. If only one typing method was used the highest discriminatory power among the S. epidermidis isolates was obtained with the lectin-binding assay which allowed 49 different strains to be detected. If the lectin-binding assay was combined with plasmid-profile analysis, all 64 different strains could be identified. The typability of lectin-binding assay was 96.9% among the S. epidermidis isolates and 25 different lectin-binding patterns were established among the 64 strains. The highest number of strains belonging to one lectin-binding pattern was 13 (20.3%). The assay was reproducible, easy to perform, relatively inexpensive and therefore applicable to large scale typing of S. epidermidis.

Bacterial Typing Techniques

Participant responses to blood gas proficiency testing reports.

There is scant objective information regarding the effect of proficiency testing for blood gases or other analytes on characterizing poor or good performers or changing laboratory behavior. Following the institution of a percentile ranking system for participants in the American Thoracic Society--California Thoracic Society Blood Gas Proficiency Testing Programs, a telephone survey disclosed characteristics of three equally sized groups of participants as follows: (1) FAIL (failed rating); (2) LOW (lowest percentile rankings without a failed rating); and HIGH (highest percentile rankings). The FAIL and LOW groups often had isolated instruments, instruments which were infrequently operated, and/or instruments operated by nontechnicians, whereas these characteristics were not found in the HIGH group. Those in the LOW group who understood their ranking and those in the FAIL group always found defects or errors and took some corrective action. This study appears to show that it is harder to maintain good performance with isolated, underutilized, or nontechnician-operated blood gas instruments, and blood gas proficiency testing is economical and valuable in assessing performance and changing behavior.

Blood Gas Analysis

Neutralizing antibodies against two HIV-1 strains in consecutively collected serum samples: cross neutralization and association to HIV-1 related disease.

97 sera collected during a 10-year period from 10 HIV-1 infected individuals were tested for neutralizing capacity against a virus isolate FICPH-22 obtained from a Danish AIDS patient, and the laboratory strain HTLV-IIIB. Three patterns of serum neutralizing activity were demonstrated: (a) patients developing high neutralizing activity against both HIV strains; (b) patients developing high neutralizing activity against the Danish virus isolate; and (c) patients developing only low titers of neutralizing antibodies (NA) against both HIV strains. The HTLV-IIIB strain was less sensitive to serum neutralization than the FICPH-22 isolate and the appearance of NA against HTLV-IIIB was typically lacking several years behind that against FICPH-22 indicating a broadening of the NA response over time. No difference in clinical outcome was observed comparing patients reaching high titers of NA and patients with low titers. Development of AIDS among patients reaching high titers of NA was preceded by a decline in NA titers, indicating an association of high titers of NA with the healthy carrier state and of declining or low titers of NA with disease progression. The majority of the neutralizing activity was mediated by IgG, but some neutralizing activity was demonstrated in the IgG depleted serum, indicating the presence of additional neutralizing substances in serum.

Acquired Immunodeficiency Syndrome

Synergistic antiviral effect in vitro of azidothymidine and amphotericin B methyl ester in combination on HIV infection.

The nucleoside analogue azidothymidine (AZT) and the methyl ester of amphotericin B (AME) were assayed for antiviral effect on HIV infection singly and in combination. Both compounds were effective in inhibiting HIV infection of MT-4 cells. At concentrations where either compound alone had no significant effect on infection, the compounds in combination were potent inhibitors of HIV as evaluated by reduction in HIV antigen production and HIV induced cytopathic effect. These results indicate that a combination therapy employing compounds with different modes of action like AZT and AME may have synergistic antiviral properties. Amphotericin B itself significantly reduced HIV infectivity in vitro and should not be used as an antifungal agent in cultures intended to propagate HIV.

Acquired Immunodeficiency Syndrome

Carbohydrates of human immunodeficiency virus.

Elucidation of the mechanism by which viral infection induces the appearance of carbohydrate neoantigens is highly important. Results from such studies could be expected to be significant for a general understanding of the regulation of glycosylation, and perhaps especially important for the understanding of glycosylation in cancer. For anti-viral therapy in AIDS, inhibitors of glycosylation enzymes are very promising as their mode of action may preclude evolvement of resistent HIV substrains, which seems to be a common problem with the reverse transcriptase inhibitors presently used. Successful therapy with glycosylation enzyme inhibitors will, however, require the development of more specific and less toxic compounds. If carbohydrate antigens can elicit a neutralizing immune response in vivo, the possibility exists that carbohydrate neoantigens can be utilized in the construction of a vaccine against AIDS.

Carbohydrate Sequence

Autologous HIV-1 neutralizing antibodies: emergence of neutralization-resistant escape virus and subsequent development of escape virus neutralizing antibodies.

The capacity of consecutive human sera to neutralize sequentially obtained autologous virus isolates was studied. HIV-1 was isolated three times over a 48-164-week period from three individuals immediately after seroconversion and from two individuals in later stages of infection. Development of neutralizing antibodies to the primary virus isolates was detected 13-45 weeks after seroconversion. Emergence of escape virus with reduced sensitivity to neutralization by autologous sera was demonstrated. The patients subsequently developed neutralizing antibodies against the escape virus but after a delay. Titers of neutralizing antibodies against late virus isolates were generally low compared to initial neutralizing titers against primary virus isolates. The delay in appearance of neutralizing antibodies to the dominant viral strain at any time in the patient and the emergence of neutralization resistant escape virus may be part of the explanation of the apparent failure of the immune system to control HIV infection.

HIV Antibodies

Effect of anti-carbohydrate antibodies on HIV infection in a monocytic cell line (U937).

Monoclonal antibodies (mAbs) against carbohydrate epitopes of gp120 have recently been found to inhibit HIV infection of lymphocytes in vitro thereby opening new possibilities for vaccine considerations. Antibody-dependent enhancement of infection has however come increasingly into focus. This study therefore investigated the neutralization of HIV in a monocytic cell line (U937) using mAbs against these carbohydrate gp120-epitopes. While antibodies against one of the epitopes (AI) neutralized infection of U937 cells despite binding to the Fc-receptor, one mAb against the sialosyl-Tn epitope enhanced infection. This enhancement was independent of complement and could be blocked by mAb Leu3a against the CD4-receptor. The study indicated that enhancement of infection in monocytic cells can occur by the same anti-carbohydrate antibodies that neutralize infection in lymphocytes, and that antibody mediated enhancement may depend on location of the epitope on gp120 rather than whether the antibody binds Fc-receptors.

Antibodies, Monoclonal

The thyroid function and size in healthy man during 3 weeks treatment with beta-adrenoceptor-antagonists.

The influence of beta-adrenoceptor antagonists on serum TSH level (supersensitive method) and thyroid volume has not previously been studied. Thirty-two young non-smoking males were treated for 3 weeks with either atenolol 50 mg (b.i.d.), metoprolol 100 mg (b.i.d.) or propranolol 80 mg (b.i.d.) in a placebo controlled study. After 1 week, median serum TSH level increased in the atenolol (from 1.76 (range: 0.96-4.04) to 2.25 (range: 1.11-4.22) mU/l, P less than 0.05) and propranolol (from 1.91 (range: 0.90-3.83) to 2.44 (range: 0.75-6.30) mU/l, P less than 0.05) treated groups. After 3 weeks, median serum TSH reached pretreatment level in the atenolol treated, whereas median serum TSH decreased compared to pretreatment values in the propranolol treated (1.68 (range: 0.68-3.62) mU/l, P less than 0.05). Except for a slight increase in the atenolol treated group, no changes in median thyroid volume was seen after 3 weeks. The changes in serum TSH or thyroid volume were not related to changes in the concentrations of thyroid hormones, or of a magnitude likely to interfere with the clinical evaluation of thyroid function.

Adrenergic beta-Antagonists

Antibody to histo-blood group A antigen neutralizes HIV produced by lymphocytes from blood group A donors but not from blood group B or O donors.

Three virus isolates HTLV-IIIB/lyA, HTLV-IIIB/lyB and HTLV-IIIB/lyO, obtained by passaging and propagating the HTLV-IIIB/H9 isolate in three separate cultures of mixed peripheral blood mononuclear cells (PBMC) from donors of blood type A, B or O, respectively, were tested for susceptibility for virus neutralization by the monoclonal antibody (MAb) AH16 directed against the blood group A epitope. MAb AH16 was previously shown to inhibit cell-free virus infection using HTLV-IIIB propagated in H9 cells. AH16 showed a concentration-dependent inhibition of the HTLV-IIIB/lyA isolate but did not inhibit the HTLV-IIIB/lyB or the HTLV-IIIB/lyO isolate. Specificity of the MAb-mediated inhibition was shown using A-antigen (tetrasaccharide). Thus, HIV infection of PBMC from donors with blood type A appears to induce expression of host-cell-encoded carbohydrate blood group A epitope on HIV which can be a target for MAb-mediated virus neutralization.

ABO Blood-Group System

Detection of human immunodeficiency virus DNA in cultured human glial cells by means of the polymerase chain reaction.

This report describes the use of the polymerase chain reaction (PCR) for the detection of viral genomic sequences in latently infected cells. Infection with human immunodeficiency virus in cultures of human glial cells was demonstrated, using nucleic acid amplification followed by dot blot hybridization. It was not possible to detect any viral antigen production in the cultures, and attempts to recover virus by highly sensitive coculture techniques were unsuccessful, indicating that the infection was latent. The PCR technique provides a simple approach to the study of viral infection in cases where viral replication is absent, or where genomic copies are present at such low numbers that they are otherwise undetectable.

Brain Neoplasms

Broadly neutralizing antibodies targeted to mucin-type carbohydrate epitopes of human immunodeficiency virus.

The cancer-related mucin-type carbohydrate neoantigen Tn was found on gp160 and gp120 of human immunodeficiency virus (HIV). Immunoglobulin G (IgG) and IgM monoclonal antibodies (MAbs) against Tn neutralized infection with cell-free virus and blocked fusion between HIV-infected and uninfected cells. This inhibition was found in infection of both lymphocytic cells and monocytoid cells. Viruses tested included six HIV-1 and five HIV-2 isolates propagated in different cells, as well as infectious plasma from AIDS patients. The antiviral effect of anti-Tn MAbs occurred by specific binding of the MAb to the virus; this binding was inhibitable by pure Tn antigen, and indications were found that this inhibition occurred at a pre-entry step. Boosting the naturally occurring low-titer anti-Tn activity may be of prophylactic value, as suggested by the in vitro neutralization found in this study.

Antibodies, Monoclonal

Acupuncture in the treatment of Graves' ophthalmopathy. A blinded randomized study.

Seventeen patients with Graves' ophthalmopathy, all euthyroid for at least one year, were included in a blinded trial to test the effect of acupuncture twice a week during two months on the eye disease, assessed by an ophthalmologist and computed tomography of the eye muscle volume. No significant change was found in eye muscle volume, Hertel measure, palpebral aperture, intraocular pressure, Hess chart, nor was there any statistically significant improvement of the irritative conjunctival symptoms.

Acupuncture Therapy

Involvement of lymphocyte function-associated antigen-1 (LFA-1) in HIV infection: inhibition by monoclonal antibody.

Monoclonal antibodies (MAbs) against the alpha- and beta-chain of lymphocyte-associated antigen-1 (LFA-1) were examined for inhibition of HIV-1 infection in vitro. Infection of the T cell line MT4 and the monocytic cell line U937 by isolates HTLVIIIB and SSI-002, respectively was inhibited in a concentration dependent manner by MAb against the beta-chain but not against the alpha-chain. No cross-reactivity was found between MAb against LFA-1 and against the CD4 receptor (MAb Leu3a). MAbs against the beta-chain and the CD4 receptor were found to act synergistically in inhibiting HIV infection. These data indicate that the beta-chain of LFA-1 in addition to the CD4 receptor may be involved in HIV infection in vitro.

Antibodies, Monoclonal