Biomedical subjects

M Ohlin

Publications and source records attributed to M Ohlin.

At least 55 records · Page 3Linked to original sources

PubMed1992

Epstein-Barr virus-induced transformation of human B lymphocytes: the effect of L-leucyl-L-leucine methyl ester on inhibitory T cell populations.

Epstein-Barr virus-mediated transformation of human B lymphocytes is inhibited by human T lymphocytes as well as by interferon-gamma. Removal of the inhibitory cell populations is essential in order to achieve successful transformation in vitro. Cells with the capacity to inhibit outgrowth of lymphoblastoid cell lines can be removed by pretreatment of peripheral blood mononuclear cells with L-leucyl-L-leucine methyl ester. This treatment eliminates monocytes, NK-cells and a CD8+ T cell subpopulation. We now show that such treatment also has toxic effects on other human T cell populations. In addition, CD4+ and/or CD8+ lymphocytes are demonstrated to contain effector cell activities which inhibit outgrowth of EBV-transformed B cells. This inhibitory activity is abolished after treatment of peripheral blood mononuclear cells or purified CD4+ T cells with L-leucyl-L-leucine methyl ester. No evidence was found for a selective toxicity against any subset within the CD4+ or CD8+ T cell populations. However, the capacity of the treated cells, both peripheral blood mononuclear cells and purified CD4+ T lymphocytes, to produce mRNA encoding IFN-gamma, a protein previously shown to downregulate outgrowth of EBV-transformed B cells, was selectively impaired. The results obtained suggest a role for CD4+ T cells to inhibit EBV-induced transformation of B cells.

Antibodies, Monoclonal↗

PubMed1992

Kinetic analysis of recombinant antibody-antigen interactions: relation between structural domains and antigen binding.

The relation between domain structures of recombinant monoclonal antibody fragments and their reaction kinetics was studied for the first time using a novel biosensor based on surface plasmon resonance technology. The association and dissociation rate constants of Fab, Fv and single domain (VH fragment) anti-lysozyme antibodies were determined and compared to the intact monoclonal antibody. Fab and Fv fragments showed similar reaction kinetics and had affinity constants of 6 x 10(9) M-1 and 25 x 10(9) M-1, respectively. The single domain antibody had significantly different reaction kinetics compared to the fragments consisting of paired heavy and light chain domains. The VH domain had both a higher dissociation and a lower association rate constant, which resulted in an affinity constant approximately 250 times lower than the Fab fragment. This rapid evaluation of antibody reaction kinetics should prove to be an important selection parameter when comparing antibody fragments for their utility in therapeutic or other applications.

Antibodies, Monoclonal↗

PubMed1992

Human monoclonal antibodies against an epitope on the class 5c outer membrane protein common to many pathogenic strains of Neisseria meningitidis.

Neisseria meningitidis is a causative agent of meningitis. Despite vaccination programs, it still causes a large number of deaths in young children. Early diagnosis followed by passive immunization with human monoclonal antibodies could be an approach to effective therapy. Peripheral blood lymphocytes from normal, healthy blood donors and from vaccinated individuals were immunized in vitro, using outer membrane proteins purified from N. meningitidis B:4:P1.15. The immunized human B cells were Epstein-Barr virus transformed and fused to a heteromyeloma. Several stable human hybridoma cell lines were established and two, secreting antibodies against the 31-kDa class 5c outer membrane protein, were characterized further. The human antibodies were of IgG1 and IgG3 isotypes, with kappa light chains. The recognized epitope was commonly found among pathogenic strains of N. meningitidis; thus, these human monoclonal antibodies may be important in the evaluation of N. meningitidis infections.

Animals↗

PubMed1992

Human MoAbs produced from normal, HIV-1-negative donors and specific for glycoprotein gp120 of the HIV-1 envelope.

Human MoAbs of IgM class were developed against three regions of the HIV-1 envelope. Uninfected donor lymphocytes were immunized in vitro with recombinant protein pB1. Four out of five antibodies were directed to different parts of the V3 region, which contains a major neutralizing site. Two out of these antibodies were directed to more than one amino acid sequence, indicating reactivity to discontinuous sites. Two of the human MoAbs inhibited viral spread between cells in tissue culture, interpreted as reactivities to conserved amino acid sequences exposed during viral maturation. No MoAb neutralized virus, which may be explained by the relatively low avidity of the antibodies. One MoAb was directed to a region containing amino acids participating in CD4 binding. This technique appears to allow formation of antibodies with fine specificities other than those obtained in infected hosts.

Amino Acid Sequence↗

PubMed1992

Real time analysis of antibody-antigen reaction kinetics.

Surface plasmon resonance, i.e. detection of changes in refractive index on a surface, was used in a biosensor to evaluate the dissociation/association rate and affinity constants of human monoclonal IgG and IgM antibodies and Fab fragments. The results showed that an observed difference in affinity constants between intact and fragmented IgG anti-tetanus antibody was related to approximately 10-fold differences in dissociation rate constants, since the association rate constants were in the same range, i.e. 2-3 x 10(5) (M-1 s-1). Affinity constants, as determined by conventional solid phase enzyme immunoassay, were substantially higher than the constants produced by the biosensor. Human monoclonal IgM anti-Tn alpha antibodies showed, furthermore, one order of magnitude higher association rate constants, as compared with the IgG antibodies, but since the dissociation rate constants were more than ten times higher, the resulting affinity constants of the anti-carbohydrate IgM antibodies were still somewhat lower than those of the IgG antibodies.

Antibodies, Monoclonal↗

PubMed1992

Evaluation of the degradation of desamino1,D-arginine8-vasopressin by nasal mucosa.

The nasal route is a convenient and simple way to administer peptides to humans. Absorption of the drug, however, requires passage of the substance through the nasal mucosa. This is a possible site of enzymatic degradation of the peptide. It is shown that rabbit nasal mucosa homogenates rapidly degrade the synthetic anti-diuretic hormone analogue desamino1,D-arginine8-vasopressin in vitro. The metabolite formed has been identified as des-(amino,arginine8,glycineamide9)- vasopressin, which is stable under the prevalent in vitro incubation conditions. It is proposed that this process is catalyzed by intracellular post-proline cleavage enzyme. Reversed phase chromatography in combination with immunological detection has been used to study the possible presence of this metabolite in the circulation after intranasal administration to humans. The metabolite des-(amino,arginine8,glycineamide9)-vasopressin could not be detected in plasma following intranasal administration, possibly indicating a paracellular absorption of desamino1,D-arginine8-vasopressin or absence of this enzymatic activity in humans.

Administration, Intranasal↗

PubMed1991

Differences in transport rate of oxytocin and vasopressin analogues across proximal and distal isolated segments of the small intestine of the rat.

The transmural intestinal passage of some oxytocin and vasopressin analogues (oxytocin, OT; [Mpa1, D-Arg8]vasopressin, dDAVP; [Mpa1, Tyr (OMe)2, carba6]oxytocin, carbetocin; [Mpa1, D-Tyr (OEt)2, Thr4, Orn8]vasotocin, antocin II; [Mpa1, D-Tyr (OEt)2, Thr4, desPro7Orn8Gly9NH2]tocinoic acid-NH(CH2)3NH2, desPOG-antocin II-NH(CH2)3NH2) was studied using isolated proximal and distal segments in the rat. All peptides (measured as peptide-like immunoreactivity) displayed a higher transport rate across distal intestinal segments as determined by radioimmunoassay (RIA). The smallest peptide, des POG-antocin II-NH(CH2)3NH2, was transported at the fastest rate. No correlation of lipophilicity with transport rate was observed. Determination of the amount of peptide remaining in the mucosal media at the end of the incubation period by HPLC did not reveal any visible degradation products. However, the large difference in transport rate between [3H]OT and immunoreactive OT indicates mucosal metabolism of this peptide. [3H]d-DAVP was distributed in a larger mucosal volume than the extracellular space marker [3H]inulin, indicating tissue uptake, but was too low (less than 100% of buffer concentration) to make an active transport mechanism likely. The differences in peptide transport rates between proximal and distal intestinal segments are most likely due to a higher distal paracellular permeability despite a decreased absorptive surface area at this region.

Amino Acid Sequence↗

PubMed1991

Production of human monoclonal antibodies in dialysis tubing.

Human x mouse hybridoma cells were grown in dialysis tubing (DT) to obtain large quantities of human monoclonal antibodies (MAb). Hybridoma cells were grown inside the DT, which was placed in a tissue culture flask containing medium. The medium inside the DT was supplemented with different additives which may be selected depending on the intended use of the MAb. About 10-50 times higher concentrations of immunoglobulin (Ig) were obtained after cultivation in DT compared to conventional tissue culture (CTC) for 2 days. The purity of the MAb was high which facilitated further antibody purification. Production of human MAb in DT proved to be excellent for evaluation studies in laboratory scale. It does not require expensive equipment and several hybridomas can be grown simultaneously.

Animals↗

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

Human monoclonal antibodies with different fine specificity for digoxin derivatives: cloning of heavy and light chain variable region sequences.

Human-mouse hybridoma cell lines producing human monoclonal antibodies against the cardiac glycoside digoxin were established after in vitro immunization or direct immortalization of human peripheral blood lymphocytes with digoxin. Three antibodies, designated MO6, LH92 and LH1114, displayed different patterns of fine specificity against digoxin and several digoxin analogues, as elucidated by inhibition ELISA. All three monoclonal antibodies had mu heavy chains, two of them (MO6 and LH114) had kappa light chains and one (LH92) lambda light chains. DNA encoding the variable regions of both heavy and light chains of the three antibodies were amplified from cDNA using the polymerase chain reaction (PCR). The nucleotide sequences of the amplified DNA were determined after subcloning of PCR fragments in M13 vectors. The deduced amino acid sequences revealed considerable sequence differences in the complementarity determining regions between the three antibodies.

Amino Acid Sequence↗

PubMed1990

Systemic and intraocular uptake of spantide, a tachykinin antagonist, following topical application to the rabbit eye.

Previous observations have indicated that topical application to the rabbit eye of tachykinin antagonists, including spantide, effectively prevents the miosis and the disruption of the blood-aqueous barrier consequent to ocular injury. The present study shows that spantide is taken up into the rabbit eye following topical application. This was established by determination of spantide in the aqueous humor by radioimmunoassay. The concentrations reached in the aqueous humor were those that could be expected to block tachykinin receptors. The elimination of spantide from the aqueous humor was found to be slow. From HPLC analysis it seemed that spantide in the aqueous humor is degraded to smaller products, predominantly spantide 5-11. Some of the topically applied peptide appeared in the general circulation. Here the rate of elimination was rapid by comparison. Very small amounts of spantide appeared in the cerebrospinal fluid after intravenous injection.

Administration, Topical↗

PubMed1990

Effects of intranigral substance P and neurokinin A on striatal dopamine release--I. Interactions with substance P antagonists.

The functional role of striatonigral neurokinins were studied by analysing the effects of intranigral injections of substance P and neurokinin A on the extracellular level of dopamine and dihydroxyphenylacetic acid in the striatum, as measured by in vivo microdialysis in rats. Two substance P antagonists, substance P D-Pro2 D-Trp7,9 and substance P D-Arg1 D-Trp7,9 Leu11 were tested and analysed for their ability to block the neurokinin effects. Unilateral injections of substance P (0.00007-7.0 nmol injected in 0.2 microliter) as well as neurokinin A (0.009-9.0 nmol) into the substantia nigra, pars reticulata of halothane anaesthetized rats produced long-lasting increases in ipsilateral striatal dopamine and dihydroxyphenylacetic acid levels. The dose-response relationship for substance P on dopamine was biphasic, with maximal effects occurring after the middle dose (0.007-0.07 nmol). The dose-response relationship for neurokinin A was monophasic. Intranigral injections of substance P D-Pro2 D-Trp7,9 (0.07-0.7 nmol) or substance P D-Arg1 D-Trp7,9 Leu11 (0.07-0.7 nmol) produced a decrease in striatal dopamine, but an increase in striatal dihydroxyphenylacetic acid. At a low dose (0.07 nmol) substance P D-Pro2 D-Trp7,9 enhanced the dopamine increase produced by intranigral substance P (0.07 nmol) or neurokinin A (0.09), while at a high dose (0.7 nmol) it blocked both substance P and neurokinin A effects. Both doses of substance P D-Arg1 D-Trp7,9 Leu11 (0.07 and 0.7 nmol) blocked the substance P- but not the neurokinin A-induced increase in striatal dopamine. Immunohistochemical analysis revealed that high doses of substance P (7.0 nmol) and neurokinin A (0.9 and 9.0 nmol), as well as substance P D-Pro2 D-Trp7,9 and substance P D-Arg1 D-Trp7,9 Leu11 (0.07 and 0.7 nmol), induced a restricted loss of tyrosine hydroxylase in dendrites and cells, and neuropeptide K in terminals, at the site of injection. Further analysis shows that co-administration of substance P (0.07 nmol) or neurokinin A (0.09 nmol) did not modify the extent of the depletion of both immunoreactivities induced by substance P D-Arg1 D-Trp7,9 Leu11 (0.7 nmol). The extent of the effect produced by substance P D-Arg1 D-Trp7,9 Leu11 (0.7 nmol) was, however, smaller than the spread of intranigral injection of [125I]Bolton-Hunter-labelled substance P D-Arg1 D-Trp7,9 Leu11, and it is suggested that the "neurotoxic" effects of the substance P antagonists are not primarily involved in their abilities to inhibit striatal dopamine release and block the stimulation of dopamine after intranigral substance P and neurokinin A.(ABSTRACT TRUNCATED AT 400 WORDS)

3,4-Dihydroxyphenylacetic Acid↗

PubMed1990

Pharmacokinetics of terlipressin after single i.v. doses to healthy volunteers.

The pharmacokinetics of triglycyl-lysine-vasopressin (TGLVP) were studied in healthy male volunteers after single i.v. injections of 5, 10 and 20 micrograms/kg b. wt. The half-life of distribution and elimination was 8 and 50 min, respectively. The volume of distribution was 0.7 l/kg b.wt. and the plasma clearance 9 ml/kg b.wt./min. These values are different from those for arginine-vasopressin and lysine-vasopressin (LVP) but confirm to some extent earlier results on TGLVP. No dose-dependent changes of the pharmacokinetics of TGLVP were evident. The LVP formation after TGLVP is described in principle using a combination of pharmacokinetic and pharmacodynamic data. Therapeutically the results in this study suggest a 4-hour interval between injections.

Adult↗

PubMed1989

Effect of viscosity on the pharmacokinetics and biological response to intranasal desmopressin.

The effect of viscosity on the nasal absorption and biological response to desmopressin was studied in humans. Nasal solutions of desmopressin with and without the addition of 0.25% (w/v) methylcellulose were administered by a precompression nasal spray pump to 10 volunteers. Plasma levels of desmopressin were assayed by radioimmunoassay and the biological response was measured by determination of the antihemophilia factors (Factor VIII and von Willebrand factor). The results showed that the addition of methylcellulose produced a more sustained and slower absorption, with a longer time to maximum plasma concentration (tmax). However, the areas under the plasma concentration-time curve were not different, indicating a similar total bioavailability. The biological response showed a similar effect. Peak Factor VIII activity was not different, but the presence of methylcellulose produced a slower onset of activity. These findings indicate that although the addition of a viscous agent to nasal formulations may produce a more sustained effect, it delays the onset of activity and no enhancement is achieved in the total bioavailability.

Administration, Intranasal↗

PubMed1989

Human monoclonal antibodies against a recombinant HIV envelope antigen produced by primary in vitro immunization. Characterization and epitope mapping.

Peripheral blood lymphocytes from healthy, HIV sero-negative blood donors have been in vitro immunized using penv9, a recombinant fragment of the envelope of HIV-1. This primary in vitro immunization followed by Epstein-Barr virus (EBV) transformation and somatic cell fusion subsequently gave rise to several specific anti-penv9 monoclonal antibodies (MO28, MO30 and MO43) of mu isotype. The hybridomas have been kept in culture for over 6 months and the antibody productivity for MO30 was measured to 18 micrograms x (24 hr x 10(6) cells)-1. The fine specificity of the antibodies was mapped by a peptide inhibition enzyme immunoassay, using overlapping synthetic pentadeca peptides covering the whole penv9. These human monoclonal antibodies exhibited a similar epitope specificity directed against a non-sequential determinant, including the amino acids 632-646, 677-681 and 687-691. This specificity is very rarely found in immune sera from seropositive patients and presently not reported in human monoclonal antibodies derived from in vivo immunized individuals, indicating that different antibody specificities can be obtained by the in vitro immunization technology. These human monoclonal antibodies did not neutralize HIV. The results presented here demonstrate the feasability of generating human monoclonal antibodies against HIV by primary in vitro immunizations, thereby avoiding the use of lymphocytes derived from infected patients when human monoclonal antibodies for therapeutic purposes are to be produced.

Antibodies, Monoclonal↗

PubMed1989

The effect of leucyl-leucine methyl ester on proliferation and Ig secretion of EBV-transformed human B lymphocytes.

The selective cytotoxicity of the lysosomotropic methyl esters of leucine or its lysosomal condensation product leucyl-leucine has been used to investigate the effect of cytolytic cells on the clonal outgrowth, cellular proliferation and antibody secretion of Epstein-Barr virus (EBV)-transformed human B cells. Large granular lymphocytes (LGL), monocytes, and a subset of T cells (CD8/CD11+) were permanently eliminated by the ester treatment. These lysosome-rich cells severely inhibit the clonal outgrowth of EBV-infected B cells, as determined by Poisson distribution calculations. Furthermore, leucyl-leucine methyl ester-treated and EBV-infected lymphocytes showed a significant increase in proliferative capability as well as immunoglobulin (Ig) production (three to 11 times) compared to non-treated but similarly infected lymphocytes. Since the effect of leucyl-leucine methyl ester treatment was also detectable in low-density (100 B cells/well) cultures, the suppression was unlikely to be exerted by EBV-specific T-cell clones, but pointed rather to the natural killer (NK) cells as effectors.

B-Lymphocytes↗

PubMed1988

Effects of concentration and volume on nasal bioavailability and biological response to desmopressin.

The effects of concentration and dose volume on the nasal bioavailability and biological response to desmopressin [DDAVP; 1-(3-mercaptoproprionic acid)-8-D-arginine vasopressin] were investigated in humans. A nasal formulation of 300 micrograms of desmopressin was administered using a premetered spray device in doses of either 1 x 50-, 2 x 50-, or 1 x 100- microL actuations to both nostrils. Intravenous administration of 0.2 micrograms/kg was also given as a reference for bioavailability calculations. Plasma levels of desmopressin were measured by radioimmunoassay. The biological response was determined by measuring circulating levels of Factor VIII (F VIII), the antihemophilia factor. Peak plasma levels of desmopressin were greatest after the 2 x 50-microL dose, followed by the 1 x 50- and 1 x 100-microL doses. The bioavailability of desmopressin from the 2 x 50-microL dose was 20%, which was significantly greater than the 11% after the 1 x 50-microL (p less than 0.01) and 9% after the 1 x 100-microL (p less than 0.001) doses. The biological response was clearly enhanced after the 2 x 50-microL dose compared with the 1 x 50- and 1 x 100-microL doses. The interindividual response in F VIII levels to nasal desmopressin ranged from 20 (CV) to 30%, which compared favorably with the 36% variation after intravenous administration. This study confirms the premetered spray device as the preferred intranasal drug delivery system, and shows that by optimizing concentration, volume, and technique of administration, a significant enhancement can be obtained in bioavailability and clinical efficacy.

Administration, Intranasal↗

PubMed1988

Development of an immunoassay for glypressin, an N-terminal extended vasopressin analogue.

The development and evaluation of a radioimmunoassay for N alpha-tri-glycyl-lysine8-vasopressin is described. The site of hapten conjugation of the immunogen has been controlled and the use of various radiolabelled tracers has been evaluated with special reference to the site of iodination. The most extensively studied antiserum showed specificity for the N-terminal triglycyl-extension as well as for several amino acid residues of the vasopressin ring. It crossreacted 27%, 28%, and 0.3% with Lys8-vasopressin, arg8-vasopressin and oxytocin respectively, and it was used to quantify triglycyl-lysine8-vasopressin in human plasma after SepPak C18 extraction. The sensitivity of the assay was 5 pg/tube with an intra-assay CV of 5-6% at 17 and 70 pg/tube. The identity of the immunoreactivity was studied by reversed phase chromatography.

Animals↗