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Biomedical subjects

B Wahren

Publications and source records attributed to B Wahren.

At least 199 records · Page 11Linked to original sources
PubMed1991

Rapid "tea-bag" peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids applied for antigenic mapping of viral proteins.

The role of individual amino acids in binding human and macaque antibodies were determined in the human immunodeficiency virus type 1 (HIV-1) gp41, residues 594-613, and for human antibodies in the hepatitis B (HB) virus core/e antigens (HBc/eAg), residues 121-140. Decapeptides with 9 amino acids (aa) overlap were synthesised using a rapid method for simultaneous multiple peptide synthesis with 9-fluorenylmethoxycarbonyl (Fmoc) protection for the alpha-amino group of the aas. One coupling cycle including washing steps was performed within 60-90 min. The crude products were analysed by reversed-phase HPLC and PD-mass spectrometry. With the 11 decapeptides covering residues 594-613 of HIV-1 gp41, the sequences SGKLI at aa 599-603 was found to be the main recognition site for 19 human anti-HIV positive sera. Two macaques repeatedly immunized with a peptide covering aa 594-613 of gp41, preferentially recognised the sequence CTTAVPW at residues 604-610 after 1-2 months of immunisation. One macaque also recognised the sequence CSGKLI, with sera sampled greater than 10 months after start of immunisation. Out of 9 human sera from patients with chronic HB, and reactive to a peptide covering residues 121-140 of HBc/eAg, 8 were found to recognise the sequence TPPA at residues 128-131, with an individual variation within residues 125-133 in regard to N- and C-terminal ends of the recognised antigenic site. Thus, human recognition of this antigenic site overlaps the reported T- and the B-cell recognition site found in mice. We believe that this simple and rapid approach to obtain large numbers of immunologically active peptides can be useful for most laboratories interested in the immunological characterisation of proteins.

Amino Acid Sequence↗
PubMed1991

Cytomegalovirus DNA detection of an immediate early protein gene with nested primer oligonucleotides.

A rapid and sensitive polymerase chain reaction (PCR) was developed to detect conserved sequences from the immediate early gene of human cytomegalovirus (HCMV). The primers sequences were from EcoRI J fragment of Ad169. The first primer set was selected to amplify a 242 bp fragment and the next primer set was nested within the first and amplified a 146 bp fragment. With the single PCR system it was possible to detect 100 fg HCMV DNA but with double PCR 5-10 fg were detectable. Specific amplification was seen in urines from patients with HCMV infections. 20 urine samples were analysed by single PCR, double PCR and virus cultivation. The double PCR was the most sensitive method. Urines from healthy seropositive persons and cells infected with other members of the herpes virus family were negative with all three methods. This suggests that specific amplification by double PCR is sensitive and can be used for rapid detection of HCMV DNA in cases with activated infection.

Antigens, Viral↗
PubMed1991

The role of alkaline phosphatase isoenzymes as tumor markers for testicular germ cell tumors.

The role of serum alkaline phosphatase as a tumor marker for testicular germ cell disease was investigated in 26 patients with testicular seminoma and 13 with nonseminomatous germ cell testis tumors. Placental alkaline phosphatase-like enzyme was elevated in 50% of the stage I seminoma patients and in all patients with stages II to III disease. In addition, liver (tissue unspecific) alkaline phosphatase was elevated in 10 and 83% of the patients, respectively. Lactic dehydrogenase and beta-human chorionic gonadotropin (beta-HCG) were detected in 50 to 60% of the patients with stage I seminoma. By combining placental alkaline phosphatase-like enzyme, lactic dehydrogenase and beta-HCG, 75% of the stage I and 100% of the stages II and III seminoma patients could be identified correctly. Placental alkaline phosphatase-like enzyme in serum also occurred with nonseminomatous germ cell tumor but less frequently, while liver alkaline phosphatase was not detected at all. Thus, placental alkaline phosphatase-like enzyme and liver alkaline phosphatase were predominantly determined in the serum of patients with seminoma. In studies of tumor tissues from 31 of these patients, those with normal serum placental alkaline phosphatase-like enzyme levels had significantly lower tissue placental alkaline phosphatase-like enzyme levels than patients with elevated serum levels (p less than 0.01). Seminoma tissues showed significantly higher levels of placental alkaline phosphatase-like enzyme and liver alkaline phosphatase than nonseminomatous germ cell tumors (p less than 0.01), explaining the infrequent elevation of serum placental alkaline phosphatase-like enzyme and liver alkaline phosphatase found in patients with nonseminomatous germ cell tumors.

Alkaline Phosphatase↗
PubMed1991

Monoclonal antibodies to conserved regions of the major core protein (gag24) of HIV-1 and HIV-2.

Five mouse monoclonal antibodies were raised against a recombinant protein comprising the complete sequence of gag24 protein from HTLV-IIIB. All monoclonal antibodies recognized the native protein in enzyme-linked immunosorbant assay (ELISA) and Western blots. All monoclonal antibodies also cross-reacted with an human immunodeficiency virus type 2 (HIV-2) strain in western blots, whereas only one antibody detected HIV-2 p25 in ELISA. By using synthetic peptides, cross-reacting epitopes were mapped and three regions were defined. The conserved immunogenic sites were located in the carboxyterminal region of the protein. Inhibition experiments with human sera showed that this region is also immunogenic in humans.

Animals↗
PubMed1991

T cell responses to peptides covering the gag p24 region of HIV-1 occur in HIV-1 seronegative individuals.

We demonstrate that peptides (16 amino acids long) covering the sequence of the HIV-1 core protein p24 induce significant proliferation in peripheral blood mononuclear cells (PBMC) of several (greater than 50%) healthy seronegative volunteers as well as seronegative homosexual men. The nature of this response was characterized and compared with those of HIV-infected patients. Several peptides induced responses; however, the most frequent responses in both seropositive and seronegative individuals were noted to the following peptides: 1 and 2 (aa 133-157); 6 and 7 (aa 183-207); 15 (aa 273-287); and 17 and 18 (aa 293-317). The response pattern was related to the disease stage of the patients; seronegative individuals as well as asymptomatic seropositive individuals (CDC II/III) responded to low concentrations of several peptides, but symptomatic patients (CDC IV) only responded to high concentrations of a few peptides. Cell separation studies of PBMC from healthy volunteers showed that the responding cells were CD4+ and expressed the CD45RO differentiation antigen. Furthermore, cord-blood mononuclear cells with less than 5% of CD45RO T cells did not proliferative to any of the peptides. Finally, CD4+ T cell lines specific for both peptides and p24 protein were successfully established from the PBMC of seronegative individuals confirming the data obtained with freshly isolated cells. These studies therefore suggest that the CD4+ cell response to p24 is not strictly disease related, instead, the response may be due to priming of the host with cross-reactive antigens.

Amino Acid Sequence↗
PubMed1991

Characterization of a major antigenic region on gp55 of human cytomegalovirus.

A major antigenic region localized to the C-terminal part of the 55K glycoprotein of human cytomegalovirus (HCMV) was mapped using synthetic peptides. Analysis of the region with six sera from healthy anti-HCMV seropositive blood donors showed that the length of the reactive sequence varied between four and eight amino acids (aa). The shortest sequence recognized was VTSG (aa 798 to 801 of the 130K precursor protein), but most sera required the three or four residues C-terminal to this to react, giving a major site of VTSGSTKD (aa 798 to 805). Using peptides immobilized on polyethylene pins, the importance of both interior (between aa essential for the binding of an antibody) and extension spacer residues was evident. Free peptides containing the reactive sequence were prepared for use in a conventional ELISA and optimal pH conditions for coating were determined. The best results were achieved at pH 2 to 3, which is in agreement with the advantageous net charge of these peptides at this low pH. Anti-HCMV positive sera showed a sensitivity of approximately 50% for both peptides on polyethylene pins and peptides coated onto plates.

Amino Acid Sequence↗
PubMed1991

Immunological characterization of the human immunodeficiency virus type 1 reverse transcriptase protein by the use of monoclonal antibodies.

Eighteen monoclonal antibodies (MAbs) directed against the purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) protein were produced. The antibodies were characterized by competitive ELISAs and Western blot experiments, and with nested, nine amino acid long peptides representing the whole 560 amino acid RT protein. By ELISA, the MAbs react with a minimum of seven epitopes of the protein. Four of the epitopes were located on the N-terminal 51K subunit and the remaining three epitopes were located at the C-terminal end of the protein. Using synthetic peptides, two epitopes at the N-terminal part were located at amino acids 294 to 302 and 350 to 354, respectively, from the N-terminal start of the protein. One epitope was located at amino acids 442 to 450, just after the cleavage site between the N-terminal and C-terminal subunit at position 440. Antibodies located at amino acids 294 to 302 could inhibit the RT enzymic activity of the protein. Two other MAbs, directed at the N-terminal and C-terminal parts of the protein, could also inhibit RT activity.

Animals↗
PubMed1991

Evaluation of a synthetic-peptide enzyme-linked immunosorbent assay for immunoglobulin M to human parvovirus B19.

A synthetic peptide corresponding to a part of the virus protein 1-virus protein 2 overlapping region of human parvovirus B19 was used in an indirect enzyme-linked immunosorbent assay. Antibodies of the immunoglobulin (Ig) M class were measured in serum samples from patients with erythema infectiosum and controls. In comparison with an IgM assay using native B19 viral antigen, the peptide antigen assay was 92% sensitive and 87% specific. B19 IgM reactivities were seen in a limited number of children with other viral diseases. Specific IgM reactivities to short synthetic viral peptides have previously been reported only with Epstein-Barr virus. Since other sources of viral antigen are limited, the peptide antigen assay may be a useful alternative for the diagnosis of B19-associated disease in human beings.

Amino Acid Sequence↗
PubMed1991

Characterization of murine monoclonal antibodies directed against the core proteins of human immunodeficiency virus types 1 and 2.

Monoclonal antibodies (MAbs) raised against the core proteins of human immunodeficiency virus type 1 (HIV-1; laboratory strain HTLV-IIIB) and HIV-2 (strain ROD) were investigated in a variety of tests, e.g., enzyme-linked immunosorbent assay (ELISA), immunostaining of Western immunoblots, immunofluorescence, immunoprecipitation, and alkaline phosphatase anti-alkaline phosphatase assay. The MAbs were grouped according to their cross-reactions. Seven HIV-1-specific MAbs reacted exclusively with HIV-1, and five showed cross-reactivity with HIV-2 and simian immunodeficiency virus of macaques in ELISA. Four of the 15 MAbs against HIV-2 reacted only with the HIV-2 protein p26. Six showed cross-reactivity with HIV-1, and five showed a broad reaction with all three viruses. Overlapping 30-amino-acid-long peptides derived from the p24 protein sequence of HIV-1 were used in an epitope-mapping system. Three different immunogenic regions (A, B, and C) could be defined. Specific regions where anti-HIV-1 and -HIV-2 MAbs cross-reacted were mapped with shorter oligopeptides.

Amino Acid Sequence↗
PubMed1991

Identification and characterization of alkaline phosphatase isozymes in human colorectal adenocarcinomas.

Using alkaline phosphatase isozyme-specific immunocatalytical assays, the content of isozymes was determined in normal mucosas and adenocarcinomas from human colon or rectum. Tumor levels of both the tissue (liver)-unspecific and the placental-like alkaline phosphatase (PLAP-like) were elevated compared to normal mucosas of the same patients. Such elevations have been reported previously, particularly in seminomas and ovarian tumors. In several tumors, moreover, the intestinal isozyme was expressed in lesser amounts than in the adjacent mucosa. The present results indicate that the activation of two of the phosphatase isozymes, including expression of the typical germ cell line phosphatase (the PLAP-like isozyme), may occur even in nongonadal tumors. This may reflect an induction pattern of phosphatase isozymes, with implications for malignant transformation also in other tumors.

Adenocarcinoma↗
PubMed1991

Search for epitopic sites of antibodies to germ cell alkaline phosphatase.

This study comprises a search for epitopic sites of the germ cell alkaline phosphatase by the use of polyclonal and monoclonal antibodies. The aim of this study was to define epitopes of the germ cell alkaline phosphatase molecule by synthetic peptides representing the known sequence of the molecule. The amino acid sequences of the epitopes to which antisera react were not known. We used overlapping peptides covering the whole 532 amino acid germ cell alkaline phosphatase molecule, including the signal 19 amino acid peptide. A polyclonal, and three monoclonal, antibodies known to react equally well with placental alkaline phosphatase isozyme (PLAP), but which differ in their reactivities with the germ cell alkaline phosphatase isozyme, were used. We have revealed potential epitopic sites of the germ cell alkaline phosphatase molecule using first polyclonal anti-PLAP, followed by monoclonal antibodies for the mapping. We could also show that the antibodies exhibited higher reactivities with such sites if they were combined in a linear synthesis. This indicates that the germ cell alkaline phosphatase molecule has discontinuous epitopes.

Alkaline Phosphatase↗
PubMed1991

Virological and clinical characteristics of human herpesvirus 6.

Six distinct human herpesviruses have been identified. They include Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2), Cytomegalovirus (CMV), Varicella-zoster virus (VZV), Epstein-Barr virus and the recently described Human herpesvirus 6 (HHV-6). With the exception of HSV-2, the members of the family are ubiquitous and infect most of the human population in the first decade of life. HHV-6 possesses morphological and structural features characteristic of members from the herpesvirus family but it is both genetically and immunologically distinct from other members. The virus was first identified in 1986 by the group at the National Institutes of Health, Bethesda. Not until 1988 was the primary disease identified as that of a common childhood disease, exanthema subitum. There are independent reports on isolations of HHV-6 from the USA, Japan, U.K., Australia and Africa, demonstrating that the virus is a widespread agent. Its possible involvement in chronic fatigue disease and as a cofactor of development of AIDS are still subjects for discussion.

Herpesviridae Infections↗
PubMed1991

Specific synthetic peptides for detection of and discrimination between HIV-1 and HIV-2 infection.

A panel of highly purified synthetic oligopeptides representing defined parts of the gag and env proteins of HIV-1 and HIV-2 were used as antigens in ELISA for serodiagnosis of HIV-1 and HIV-2 infection. The analysis included sera from 321 HIV-infected patients and 201 healthy controls from the Ivory Coast, where the prevalence is high for both HIV-1 and HIV-2, and sera from European HIV-1-infected individuals. All sera from HIV-1-infected individuals reacted with a 20 amino acid (a.a.) peptide JB-4c (a.a. 594-613) derived from a highly immunogenic conserved region of the external part of gp41. An equally good response was seen in the HIV-2-infected individuals to a 20 a.a. peptide, JB-16c, from the corresponding part of HIV-2 gp36. Both HIV-1- and HIV-2-seropositive individuals responded well to a peptide, JB-8pc (a.a. 427-448), representing the C-terminal end of the putative CD4-binding site of gp120 of HIV-1. The frequency of reactivity to three selected HIV-1 gag peptides derived from p17 and p15 was 60-70% in both HIV-1 and HIV-2-positive sera. To distinguish between HIV-1 and HIV-2 infection, the sera were titrated against the peptides. Although there was a high degree of cross-reactivity at lower serum dilutions, it was possible to discriminate the infections at higher dilutions to the HIV-1 and HIV-2 gp41/gp36 peptides JB-4c and JB-16c. Analysis of serum reactivity to several selected peptides thus allowed the identification of HIV infection, and the discrimination between HIV-1 and HIV-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Africa, Western↗
PubMed1991

Characterization of human monoclonal antibodies directed against the pp65-kD matrix antigen of human cytomegalovirus.

Human monoclonal antibodies specific for human cytomegalovirus (CMV) antigens have been established using peripheral blood lymphocytes from a seropositive donor. Immortalization of antigen-specific B cells was achieved by Epstein-Barr virus transformation followed by somatic cell fusion of antigen-specific lymphoblastoid cells. Four clones producing high-affinity antibodies (0.2-7 x 10(9) M-1) specific for the viral matrix protein pp65 have been further characterized with respect to epitope specificity of secreted antibodies. The studied antigen represents a major protein produced by in vitro-cultivated virus, and is important in the serodiagnosis of CMV infection. The human monoclonal antibodies recognized different epitopes, some of which proved to be overlapping. The fine specificity of these antibodies was evaluated using synthetic peptides covering the sequence of pp65. The antibody MO58 recognized a linear epitope (residues 283-288) whereas antibody MO53 recognized a discontinuous epitope involving residues 208-216 and 280-285. Despite the close proximity of these epitopes, the antibodies did not compete with each other for the same binding site on intact antigen.

Amino Acid Sequence↗
PubMed1991

Identification of amino acids in the V3 region of gp120 critical for virus neutralization by human HIV-1-specific antibodies.

The importance of the dependence on single amino acids in the V3 region of HIV-1 gp120 was evaluated for virus neutralization and antibody-dependent cellular cytotoxicity (ADCC). Synthetic overlapping 15-mer peptides and a set of omission peptides covering amino acids 301-317 were used. Sera from 29 HIV-1-infected individuals at different stages of disease were tested for neutralization, ADCC and specific IgG reactivity. Six HIV-1 neutralizing monoclonal antibodies (mAb) acted as controls. All mAb reacted with a region (amino acids 304-318) of gp120, previously shown to induce neutralizing antibodies. The amino acids essential for reactivity were identified to be within the sequence GPGR (amino acids 312-315). The importance of this region for occurrence of neutralizing antibodies in infected humans was investigated using the same set of peptides. Out of 29 individuals, 21 were found to have neutralizing antibodies in titres between 100 and 1000. Among the neutralization-positive sera, 17/21 (81%) reacted with amino acids 304-318, compared with only one of eight sera (13%) negative in neutralization. When any of the four amino acids G, P, G or R were deleted, the seroreactivity decreased considerably. The conserved sequence GPGR was therefore considered to be the most important for neutralization in this region in human sera as well. Thus, the conserved sequence GPGR in the V3 region of gp120 is critical for virus neutralization by human HIV-1-specific antibodies.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Antiviral effects of 3'-fluorothymidine and 3'-azidothymidine in cynomolgus monkeys infected with simian immunodeficiency virus.

An acute infection with simian immunodeficiency virus (SIVSM) in cynomolgus monkeys was used to evaluate the antiviral effects of 3'-fluorothymidine (FLT) and 3'-azidothymidine [zidovudine (ZDV)]. Neither compound prevented the infection despite dosing prior to virus inoculation. FLT was about ten times more potent than ZDV in delaying the appearance of SIVSM antigen in the monkeys. The serum half-life of FLT was longer than that of ZDV and ZDV was bound to plasma proteins to about 60% while FLT was virtually unbound. It is proposed that the in vivo difference in potency between ZDV and FLT could, at least partly, be explained as the combined effects of a longer plasma half-life and a higher free concentration of FLT and possibly a higher intracellular concentration of the triphosphate of FLT.

Animals↗