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K J Krohn

Publications and source records attributed to K J Krohn.

8 recordsLinked to original sources
PubMed1992

Fine specificity of the B-cell epitopes recognized in HIV-1 NEF by human sera.

We have previously used partially overlapping synthetic nonapeptides to characterize the human natural antibody response against HIV-1 negative regulatory factor (NEF), and identified nine 5 to 13 amino acid long regions that were recognized by sera of HIV-1-infected individuals. In this report we define the minimal size of these epitopes with the use of shorter, from 3 to 8 amino acid long partially overlapping peptides covering the complete sequence of the previously identified reacting regions and the N- and C-terminal flanking sequences. We also introduce a new method for the analysis of the reactivities obtained with peptides of different lengths. In six of the antigenic regions the epitopes were found to be noncontiguous and to consist of multiple, down to three amino acid long separate reactive stretches (epitope 1: WSK, VGW, TVRERMRR; epitope 3A: PLRPM, SHFLK; epitope 3B: SQRRQD, DLW; epitope 3C: IYHT, QGYFPDWQN; epitope 4: SLL, VSL; epitope 5: EVLEWRFDSR, VAR). Three epitopes were clearly linear (epitope 2: CAWLE; epitope 3D: LTFGWC; epitope 6: PEYF). Interestingly, five of the minimized B-cell epitopes (1, 3A, 3C, 3D, 5) recognized by human sera overlap totally or partly with the previously identified T-cell epitopes in HIV-1 NEF. Also, only three of the epitopes (3C, 3D, 5) were in a computer-based homology search shown to contain strictly NEF-specific sequences.

Amino Acid Sequence↗
PubMed1992

Immunological variation and immunohistochemical localization of HIV-1 Nef demonstrated with monoclonal antibodies.

OBJECTIVE: To study the immunological and immunohistochemical nature of HIV-1 Nef. DESIGN: Monoclonal anti-Nef antibodies were generated and used to identify antigenic epitopes in Nef, to study immunological cross-reactivity between Nef from different isolates and to reveal the subcellular localization of Nef. METHODS: Monoclonal antibodies against recombinant HIV-1 Nef protein (BRU isolate) were generated in BALB/c mice. The epitope mapping was carried out with the use of overlapping 15-20mer lipopeptides linked to a lipid group at the amino-terminus. Immunoperoxidase method was used for histochemical studies. RESULTS: Ten stable antibody-producing clones, mainly of the immunoglobulin (Ig) G1 subtype, with strong Western blot and enzyme-linked immunosorbent assay reactivity toward the recombinant Nef protein, were obtained. The epitopes recognized were located on amino-acid sequences 21-41, 31-50, 51-71, 61-80, 151-170, 161-180, and 171-190. All 10 monoclonal antibodies also reacted with the native Nef of HIV-1BRU, and eight reacted with native HIV-1IIIB. Most antibodies also reacted with Nef from more divergent HIV-1 strains. In Western blotting, two forms of Nef (24 and 27 kDa) were observed with most isolates studied. Immunohistochemical staining of HIV-1-infected H9 or MT-4 lymphoid cells demonstrated that Nef was expressed mainly in the Golgi complex and at the nuclear membrane, but occasionally also in the nucleus. The nuclear localization of Nef was especially frequent in the HIV-1-infected MT-4 cells. CONCLUSIONS: Our findings suggest that Nef is expressed in two isomorphic forms, and that it may also act as a nuclear protein and thus have a direct regulatory function at the RNA/DNA level.

Animals↗
PubMed1992

Serological responses to mycoplasmas in HIV-infected and non-infected individuals.

OBJECTIVE: To assess the frequency of mycoplasma infections in HIV-antibody-positive and -negative individuals by studying the serological responses against mycoplasmas, especially Mycoplasma fermentans and M. pirum. DESIGN: An enzyme-linked immunosorbent assay (ELISA) was used to measure immunoglobulin G (IgG) class antibody concentrations against six mycoplasma species in sera of HIV-positive and HIV-negative individuals. METHODS: Serum samples were obtained from 30 HIV-positive individuals (10 asymptomatics, 10 with lymphadenopathy syndrome and 10 with AIDS), 10 HIV-negative partners of HIV-positive individuals and 40 HIV-negative blood donors. Antibodies to M. fermentans strains incognitus and PG18, M. pirum, M. genitalium, M. pneumoniae and M. hominis were assessed by immunoblot or ELISA. Absorbance values were taken as a semiquantitative measurement for antibody concentration and an arbitrary cut-off value (0.8) was set to establish seroprevalence. RESULTS: There was no significant difference in the mean IgG concentrations of any of the six mycoplasmas between HIV-positive and HIV-negative groups. Antibody concentrations were also similar in different clinical phases of HIV infection. Antibody concentrations to different mycoplasma strains were compared with each other to reveal eventual cross-reactions caused by shared antigens; the strongest correlation (r = 0.836) was found between M. fermentans strains incognitus and M. pirum antibody concentrations. The correlation between M. fermentans strains incognitus and PG18 was also significant but weaker (r = 0.522). No shared antigens between M. fermentans strain incognitus and M. pirum were demonstrated by immunoblot. CONCLUSIONS: Antibodies against M. fermentans type strain PG18, strain incognitus and against M. pirum are detected infrequently and their presence does not correlate with HIV infection per se or with the clinical stage of HIV infection.

Antibodies, Bacterial↗
PubMed1992

Cellular localization of Nef expressed in persistently HIV-1-infected low-producer astrocytes.

OBJECTIVES: The characterization and localization of HIV-1 Nef highly expressed in permanently infected astrocytes (TH4-7-5) as a model for latent infection of human brain cells. DESIGN: Immunochemical methods are an appropriate tool to investigate expression and localization of cellular proteins. METHODS: Nef expression was analysed by Western blot and immunoperoxidase staining using a panel of monoclonal and polyclonal antibodies. Cellular localization studies were performed by indirect immunofluorescence and subcellular fractionation of TH4-7-5 cells. Myristoylation of Nef was investigated by immunoprecipitation of [3H]myristic acid-labelled cell extract. TH4-7-5 nef gene was cloned and amplified by polymerase chain reaction and the nef nucleotide sequence analysed. RESULTS: Reactivities of various Nef-specific antibodies with Nef antigen in TH4-7-5 cells were demonstrated by Western blot analysis. Immunofluorescence revealed cytoplasmic perinuclear staining of Nef with most antibodies. However, one monoclonal antibody against amino acids 168-175 of Nef showed intense homogeneous nuclear staining in TH4-7-5 cells. Reactivity of this Nef antibody was blocked with recombinant Nef derived from TH4-7-5 cells. After subcellular fractionation, Nef was detected in nuclear, membrane and cytosolic fractions of TH4-7-5 cells. No myristoylated Nef antigen was detectable, perhaps because of a serine residue at position 2 of the TH4-7-5 nef gene instead of the glycine residue required for myristoylation. CONCLUSIONS: Chronically HIV-1-infected astrocytoma cells with restricted virus production express different antigenic forms of Nef, which can be distinguished by their subcellular localization. Variant subcellular targeting of Nef suggests the existence of multiple activities of Nef within HIV-infected cells.

Amino Acid Sequence↗
PubMed1989

Imprint cytology in immunocytochemical analysis of oestrogen and progesterone receptors of breast carcinoma.

Cytological imprint material from 26 mammary carcinomas was stained with monoclonal antibodies to oestrogen and progesterone receptors in an immunoperoxidase procedure. The staining result was compared with that of parallel stainings of frozen tissue sections of the same tumours. The peroxidase reactions in both techniques were semiquantitatively assessed (histoscore). In both sets of stainings the results agreed in 25 of 26 cases (oestrogen receptor: 19 positive, six negative; progesterone receptor: 14 positive, 11 negative). The histoscores of imprint preparations and cryostat sections showed a significant correlation in linear regression analysis (oestrogen receptor: r = 0.755, p less than 0.001; progesterone receptor: r = 0.740, p less than 0.001). Imprint cytology is simple, does not require expensive instruments, and no separate specimen has to be sequestered. It is especially suitable for immunocytochemical steroid receptor analysis of small breast carcinomas.

Adult↗
PubMed1979

Human transfer factor in vitro. II. Augmentation of the secretion of leucocyte migration inhibitory factor (LIF) by leucocyte dialysate and by its components L-serine and glycine.

The effect of human transfer factor (TF) or its components L-serine and/or glycine in tuberculin (PPD), or leucoagglutinin (LA) induced leucocyte migration inhibitory factor (LIF) secretion was studied. Augmentation of LIF secretion was seen with low concentration ( = 0.078 g/l) of TF when lymphocytes were cultured in minimum essential medium for suspension cultures (MEM-S), a culture medium lacking L-serine and glycine. High concentrations (0.3125-5.0 g/l dry weight) of TF were inhibitory in MEM-S. In RPMI 1640, a culture medium containing L-serine and glycine, TF was either inhibitory or had no effect. The combination of L-serine and glycine, at concentrations equivalent or lower than the optimum of TF, had an augmenting effect on LIF secretion identical to that of TF, but no inhibition at higher concentrations was seen. The results indicate that human TF contains components which have suppressive or augmenting effects on LIF secretion in vitro. The augmenting effect may be mainly due to L-serine and glycine and thus not related to TF's activity in vivo.

Cells, Cultured↗
PubMed1978

The effect of transfer factor on lymphocyte transformation. Comparison of augmentation by dialysates of leucocytes and lymphoid and non-lymphoid organs.

In this paper we describe experiments to determine whether dialysable extracts of non-lymphoid and lymphoid organs augment lymphocyte transformation in vitro in a manner similar to the augmenting activity of leucocyte dialysates. Human peripheral blood leucocytes were cultured with tuberculin PPD or leucoagglutinin, and dose-related effects of the dialysable extracts on lymphocyte transformation were studied by 125IUdR incorporation. Augmentation of lymphocyte transformation was obtained not only with leucocyte dialysates but also with dialysable extracts of lymphoid and non-lymphoid organs (e.g. spleen, liver, kidney, brain). It is concluded that the agent or agents present in dialysable leucocyte transfer factor preparations, which augment lymphoid transformation in vitro, are widely distributed throughout mammalian tissues.

Animals↗