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Biomedical subjects

K Falk

Publications and source records attributed to K Falk.

At least 55 records · Page 3Linked to original sources
PubMed1995

The role of repetitive DNA sequences in the size variation of Epstein-Barr virus (EBV) nuclear antigens, and the identification of different EBV isolates using RFLP and PCR analysis.

The six Epstein-Barr virus (EBV) nuclear antigen proteins (EBNA-1-6) show characteristic size variations between different virus isolates; this is a feature that has been used to identify the source of virus isolates in epidemiological studies (Ebnotyping). We have now studied the correlation between restriction fragment length polymorphisms (RFLPs) within exons coding for the EBNAs and the molecular masses of the respective proteins. The B95-8 EBV strain was used as the prototype virus. The variation in apparent molecular mass of EBNA-1, -3 and -6 correlated positively with the size of RFLP coding for repeat sequences in these polypeptides. For EBNA-2, no correlation between apparent molecular mass and length of the repetitive sequences was found. The EBNA-4 protein showed virtually no variation in apparent molecular mass and RFLP size across the repeat sequence. Based on the strong correlation between apparent molecular mass and RFLP size for EBNA-6, we developed an EBNA-6 PCR assay that discriminated between different isolates of EBV. This assay offers the advantage of EBV characterization using uncultured material (e.g. throat washings, blood or biopsies), thus avoiding the selection against poorly transforming strains that occurs during establishment of lymphoblastoid cell lines required for Ebnotyping at the protein level.

Animals↗
PubMed1995

Isolation of the causal virus of infectious salmon anaemia (ISA) in a long-term cell line from Atlantic salmon head kidney.

A long-term cell line (SHK-1) supporting replication of the causal virus of infectious salmon anaemia (ISA) has been established. The cell line was developed from a culture of Atlantic salmon (Salmo salar L.) head kidney cells. CPE was observed in SHK-1 cells 12-14 days after inoculation with ISA-infective tissue material. The time for CPE to develop decreased after repeated passages of medium from infected cell cultures to new cultures. Transmission trials demonstrated that Atlantic salmon parr developed ISA after intraperitoneal injection of preparations made from infected cells and growth medium. The ISA infectivity of the cell preparations increased with incubation time of inoculated cells. Cell cultures in a second passage were found to have a higher infectivity than the primary inoculated cultures. Virus particles with a diameter of approximately 100-120 nm, and which contained an external envelope and granules were seen in electron micrographs of thin sections of infected cells. Most of the virus particles were located extracellularly close to the cell surface, and in some cases, a connection between virus and plasma membrane could be observed. This indicates that virus particles were released by budding. Enveloped virus particles of 45-140 nm in diameter were seen in abundance in electron micrographs of a negatively stained purified virus preparation. Large, highly pleomorphic particles up to 700 nm in the longest dimension were occasionally observed in unpurified preparations. The evidence is therefore strong that the virus isolated in SHK-1 cells is the aetiological agent of ISA.

Anemia↗
PubMed1995

Propagation of infectious salmon anaemia (ISA) virus in cell culture.

A long-term cell line supporting growth of the infectious salmon anaemia (ISA) virus has been established. The cell line (SHK-1) was developed from a culture of head kidney leucocytes from Atlantic salmon, and exhibited macrophage-like enzyme reactivities. By means of transmission experiments, ISA infectivity of cell culture medium could be demonstrated from day 5 after infection of SHK-1 cells with ISA-infective tissue homogenate. ISA infectivity of cell culture medium increased following repeated passages of virus. ISA-infected cell cultures develop cytopathic effects (CPE), making quantitation of virus possible. The development of CPE in ISA virus infected cells was inhibited by ammonium chloride, chloroquine and bafilomycin A, suggesting that infection of SHK-1 cells with ISA virus requires a low-pH step.

Anemia↗
PubMed1995

Demonstration of infectious salmon anaemia (ISA) viral antigens in cell cultures and tissue sections.

Rabbit polyclonal antibodies and mouse monoclonal antibodies (MAb) directed against infectious salmon anaemia virus (ISAV) were produced using a virus prepared in a newly established cell line culture developed from Atlantic salmon head kidney (SHK-1) cells. These antibodies were used to establish an indirect fluorescent antibody test for the detection of viral antigens in cell cultures and tissue cryosections. Specific fluorescence was detected in ISAV-infected cell cultures using rabbit polyclonal and MAb. One selected MAb produced specific staining for ISAV in the tissue sections from all the examined organs of ISAV-infected Atlantic salmon 20 d post infection. Fluorescence was mainly found in the endothelial cells and in single cells scattered throughout the parenchyma of the organs.

Anemia↗
PubMed1994

Isolation of naturally processed peptides recognized by cytolytic T lymphocytes (CTL) on human melanoma cells in association with HLA-A2.1.

Cytolytic T lymphocyte (CTL) clones have previously been derived from peripheral blood of melanoma patient SK29(AV). They lyse autologous melanoma cells but not autologous Epstein-Barr virus (EBV)-transformed B lymphocytes. Immunoselection experiments indicate that these CTL clones recognize 4 different antigens (Aa, Ab, B, C) in association with a single HLA restriction element, HLA-A2.1. While the expression of antigens B and C appears to be confined to SK29-melanoma cells, antigens Aa and Ab are shared by a high proportion of allogeneic HLA-A2-positive melanoma lines. HLA-A2.1 and total HLA class I molecules have now been purified from SK29-melanoma cells using affinity chromatography and associated peptides have been eluted. Peptide pools eluted from HLA-A2.1 and total class I were separated by reversed phase high performance liquid chromatography (HPLC). Individual HPLC fractions were tested for their ability to sensitize target cells for recognition by SK29-CTL clones. The presence of antigens Aa, Ab, B and C was detected in distinct HPLC fractions that were identical for both peptide pools. As target for detection of peptide antigens in HPLC fractions, the use of the HLA-A2.1-positive antigen processing mutant cell line CEM x 721.174.T2 (T2), pre-incubated with anti-HLA-A2 monoclonal antibody (MAb) MA2.1, was shown to be essential. Single-peak target-sensitizing activity was found for antigens Ab and B, whereas multi-peak sensitizing activity was reproducibly detected for antigens Aa and C. We reason that at least some of these melanoma peptide antigens might occur in biochemically distinct isoforms.

Antigens, Neoplasm↗
PubMed1994

Pool sequencing of natural HLA-DR, DQ, and DP ligands reveals detailed peptide motifs, constraints of processing, and general rules.

We have approached the problem of MHC class II ligand motifs by pool sequencing natural peptides eluted from HLA-DR, DQ, and DP molecules. The results indicate surprisingly clear patterns, although not quite as clear as with natural class I ligands. The most striking feature is a highly dominant Proline at position 2. We interpret this to be a consequence of aminopeptidase N-like activity in processing. Another general aspect is the existence of three to four hydrophobic or aromatic anchors, whereby the first and the last are separated by five to eight residues. The peptide motifs for HLA-DR1, DR5, DQ7, and DPw4 are allele-specific and differ by spacing and occupancy of anchors. The anchors tend to be flanked by clusters of charged residues, and small residues, especially Ala, are frequent in the motif centers. These detailed motifs allow one to interpret most previous (DR-) motifs as fitting one or more of the anchors or conserved clusters. The relative motif symmetry suggests the possibility of bidirectional binding of peptides in the class II groove.

Alleles↗
PubMed1994

Origin, structure and motifs of naturally processed MHC class II ligands.

In the past few years a considerable number of naturally processed MHC class II ligands have been identified and sequenced. Most of them derive from endogenous sources, predominantly from plasma membrane proteins. Generally, they display variability in length but exhibit characteristic patterns of invariant amino acid positions, which reflect the allele-specific binding requirements. As a general feature, class II ligands also often contain a pattern of proline residues interpreted as a 'processing motif'.

Amino Acid Sequence↗
PubMed1994

Comparison of immunohistochemistry, acid-fast staining, and cultivation for detection of Mycobacterium paratuberculosis in goats.

Forty-seven paired specimens of ileum and mesenterial lymph nodes from goats originating from 2 herds with paratuberculosis were investigated. Culture of the specimens for Mycobacterium paratuberculosis was compared with Ziehl-Neelsen staining and with immunohistochemical tests on paraffin-embedded tissue sections, using a M. paratuberculosis immune serum and an avidin-biotin-alkaline phosphatase method. Immunohistochemical techniques detected most positive samples and produced more clearly visible reactions than did acid-fast staining. Eighteen of 47 samples were positive by immunohistochemical techniques may represent a valuable adjunct to standard techniques for diagnosis of paratuberculosis in goats.

Animals↗
PubMed1994

Analysis of a naturally occurring HLA class I-restricted viral epitope.

A previously described nonapeptide sequence motif for antigens recognized by T cells in the context of the human major histocompatibility complex (MHC) molecule HLA-A2.1 was used to identify the natural epitope of influenza A virus matrix protein. We show here that the peptide with the sequence GILGFVFTL is the synthetic analogue of the natural epitope by demonstrating the presence of the corresponding peptide on MHC molecules of virus-infected cells. The role of the hydrophobic anchor amino acids in positions 2 and 9, which constitute the epitope motif, was investigated with synthetic variants of the epitope and cytotoxic T lymphocytes as indicator cells. The crucial role of the side chains of amino acids in those positions was evidence by their influence on the efficiency of T-cell stimulation.

Amino Acid Sequence↗
PubMed1993

Allele-specific peptide ligand motifs of HLA-C molecules.

The consensus motifs of HLA-Cw3, -Cw4, -Cw6, and -Cw7 ligands were determined by pool sequencing. Together with information obtained by sequencing of some prominent individual peptides, the results indicate the following: (i) all four HLA-C molecules are associated with peptides. (ii) These peptides adhere to allele-specific motifs that are similar to those of to HLA-A or -B molecules; they have a preferred length of nine amino acids and an anchor residue at the C terminus. (iii) All four HLA-C molecules analyzed exhibit related peptide motifs, although each allelic product shows individual characteristics in fine specificity. (iv) Processing and origin of peptides appear not to be different from that of other class I molecules. (v) No obvious difference at C-terminal position 9 was present in the peptides isolated from the two dimorphic variants of HLA-C that determine dominant resistance to natural killer NK1-specific cells (HLA-Cw4, -Cw6) or to NK2-specific cells (HLA-Cw3, -Cw7) and that differ in two residues in or near the pocket at position 9.

Amino Acid Sequence↗
PubMed1993

Qa-2 molecules are peptide receptors of higher stringency than ordinary class I molecules.

Class I molecules of the major histocompatibility complex (MHC) transport peptides to the cell surface for surveillance by T cells. Ligand specificity is stringent and differs from allele to allele. Here we report analysis of natural ligands of 'unconventional' glycophosphatidyl-anchored mouse class I molecules, Qa-2. The function of these molecules is unclear; they can serve as recognition structures for 'unrestricted' cytotoxic T cells but have not been found to present peptides to T cells, although the DNA sequence suggests a similar peptide binding groove to that of 'conventional' class I molecules, and other unconventional class I molecules can present antigens in a few cases. Pool sequencing of natural Qa-2 ligands shows that Qa-2 molecules are indeed peptide receptors, having ligand specificity similar to that of conventional class I molecules, that is, a predominant length of nine amino acids, anchor positions, and hydrophobic termination of peptides. But ligand specificity is much more stringent than with other class I molecules: of the nine positions, two are anchors and four have rather limited occupancy.

Amino Acid Sequence↗
PubMed1993

Both human and mouse cells expressing H-2Kb and ovalbumin process the same peptide, SIINFEKL.

HeLa cells, derived from a human cervix carcinoma line, were transfected with a mouse MHC class I gene, H-2Kb, and chicken ovalbumin. H-2Kb-restricted cytotoxic mouse T cells specific for ovalbumin recognized the double-transfected human cells with similar efficiency as ovalbumin-transfected EL4 mouse thymoma cells (H-2b). The naturally processed ovalbumin T cell epitope was eluted from H-2Kb molecules from double-transfected HeLa cells and was biochemically compared to a synthetic peptide, SIINFEKL, known to be the natural Kb ligand of ovalbumin-transfected H-2b mouse cells. The results indicate that the ovalbumin-derived Kb-ligand of double-transfected HeLa cells is also SIINFEKL. Thus, both human cervix carcinoma cells and mouse thymoma cells expressing Kb and ovalbumin process the same octapeptide. Together with previous data, derived by comparing Kb ligands of unknown sequences from both human and mouse cells expressing Kb, it can be concluded that both mouse and human cells are capable of processing the same ligands for mouse MHC class I molecules. Hence, the general specificity of the peptide-generating mechanism for class I ligands is apparently conserved between evolutionary distant species.

Amino Acid Sequence↗
PubMed1993

Consensus motifs and peptide ligands of MHC class I molecules.

The introduction of a powerful peptide isolation method has paved the way for the characterization of the natural peptide-ligands of MHC class I molecules. More than 50 are already known by their amino acid sequence. As a striking feature, all these peptides display some common sequence characteristics: a distinct peptide length, normally 8 or 9 amino acids, and typically two invariant 'anchor' amino acids. These 'consensus-motifs' are different for each MHC-class-I allele and their usefulness for the precise prediction of peptide antigens has already been demonstrated. This review discusses the consensus motifs known at present and lists most of the sequences referring to natural MHC-ligands.

Alleles↗
PubMed1993

An origin of DNA replication (oriP) in highly methylated episomal Epstein-Barr virus DNA localizes to a 4.5-kb unmethylated region.

Epstein-Barr virus (EBV) reveals two types of interactions with human B lymphocytes. In immortalized lymphoblastoid cell lines (LCL), nine virus-coded proteins are expressed: six nuclear (EBNA1-6) and three membrane proteins (LMP1, TP 1,2). In Burkitt's lymphoma (BL) tumors and representative cell lines (type I) only EBNA-1 is expressed. The viral genomes are carried by the cells in an extrachromosal, episomal form. We have earlier described an overall difference in EBV DNA methylation between LCLs of normal B-cell origin and type I BL lines. EBV genomes are hypomethylated in the former, methylated in the latter. The origin of replication (oriP) for the episomal DNA is exempted from this overall methylation. This unmethylated region was analyzed in the BL derived type I cell line Rael. It starts just upstream of the coding exons for the small untranslated RNAs EBER I/II. It extends throughout oriP and includes several control elements between oriP and the first promoter for EBNA transcription (BCR2). The viral DNA was methylated around and downstream of that promoter, which is not used for EBNA-1 expression in Rael.

Animals↗