Biomedical subjects

L Jaso-Friedmann

Publications and source records attributed to L Jaso-Friedmann.

At least 37 records · Page 2Linked to original sources

PubMed1998

Receptor associated phosphorylation following monoclonal antibody or synthetic peptide binding to nonspecific cytotoxic cells.

We have previously shown that crosslinkage of a receptor protein on catfish nonspecific cytotoxic cells (NCC) with anti-receptor monoclonal antibody or with a synthetic peptide activates cytotoxicity and initiates signalling responses. Receptor linked signalling was associated with the production of increased levels of expression of 50-60 and 20-30 kDa phosphoproteins determined by immunoprecipitation with anti-phosphoserine and anti-phosphotyrosine mabs. These proteins are components of a macromolecular protein complex (>200 kDa) determined by reducing and nonreducing SDS-PAGE. The calcium ionophore A23187 treatment produced the same pattern of phosphoprotein expression as peptide or mab. Maximum phosphoserine expression occurred at 15'-30' post-mab binding. We now show that synthetic peptide or mab treatment initiated the same serine and tyrosine phosphorylation profiles. The PKC specific inhibitor MDL 29,152 produced 50% inhibition of NCC lysis of IM-9 target cells, and completely inhibited serine phosphorylation of peptide activated cells but had no effect on tyrosine phosphorylation of the phosphointermediates. Genistein pretreatment of NCC inhibited cytotoxicity and tyrosine phosphorylation. Sequential immunprecipitation of the phosphointermediate demonstrated that the phosphorylated serine and tyrosine residues were on the same 50-60 kDa protein. These data indicate that both proximal and distal signalling events required for NCC activation may be associated with ATPase phosphorylation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

Molecular characterization of a protozoan parasite target antigen recognized by nonspecific cytotoxic cells.

The target cell antigen(s) on tumor cells and on protozoan parasites recognized by NK and nonspecific cytotoxic cells (NCC) has not yet been specifically identified. NCC may be the teleost equivalent of NK cells and IL-2-activated NK cells. A ligand recognized by NCC has been identified. It is expressed on both protozoan parasites and mammalian tumor target cells. In the present study, a protozoan parasite antigen (NK target antigen/NKTag/p46) was purified from Tetrahymena pyriformis and the entire amino acid sequence was deduced from cDNA. Soluble and purified NKTag inhibited NCC lysis of human and mouse transformed target cells. Homology comparisons using Swissprot database revealed that NKTag is a novel protein. Molecular weight computation of the deduced sequence demonstrated that NKTag is a 48.17-kDa protein containing 422 amino acids with relatively high percentages of tyrosine and serine residues. Expression of NKTag on various mammalian tumor target cells, normal tissue, and T. pyriformis was determined using anti-multiple antigenic peptide (MAP) monoclonal antibody (mab) 22A12 [generated against an N-terminal 20-mer (aa 61-80) of p46]. This mab bound to tissue-cultured and tumor cells (YAC-1, IM-9, NC-37, MOLT-4, and U937) with low levels of binding to fish, mouse, and equine cells. Studies were also done to determine if purified and iodinated NKTag bound specifically to NCC. Binding was saturable and specific. These data provide evidence that NCC recognize a target cell ligand which is found on both protozoan and tumor cells. This may provide an explanation as to how NCC (including activated NK cells) recognize a vast array of targets in the absence of haplotype recognition and in spite of a diverse species of origin.

Amino Acid Sequence↗

PubMed1996

Activation of nonspecific cytotoxic cells with a multiple antigenic peptide: specificity and requirements for receptor crosslinkage.

Nonspecific cytotoxic cells (NCC) of teleost fish recognize a conserved antigenic determinant found on the protozoan Tetrahymena pyriformis and on many different tumor target cells. This determinant is located on a 46-kDa Tetrahymena protein referred to as natural killer target antigen (NKTag). The NKTag cognate sequence recognized by NCC is composed of seven to nine amino acids. In the present study, synthetic peptides of the cognate NKTag determinant were prepared as multiple antigenic peptides (MAP). Immobilized MAP activated NCC lysis of IM-9 target cells in the absence of antigen presenting cells or exogenous added cytokines. NCC binding to immobilized MAP produced two- to fivefold increased lysis of IM-9, U937, and HL-60 target cells compared to scrambled control MAP (composed of the same amino acids only in random sequence). NCC receptor crosslinkage was required for activation. Immobilized monomeric homologous cognate peptide did not activate increased NCC lysis of IM-9 target cells; however, NCC preincubated with soluble homologous monomer inhibited MAP activation of lysis. Ligand activation of NCC was antigen specific. Binding of the immobilized ligand with anti-MAP mab 22A12 prior to addition of NCC blocked activation. Mab 22A12 also inhibited NCC lysis of IM-9 target cells. A possible mechanism of NCC activation was determined. Binding of NCC to immobilized MAP produced significantly increased membrane expression of a putative receptor as defined by mab 5C6 binding. These studies demonstrate that activation of NCC by a target cell antigenic determinant depends on crosslinkage of an NCC "receptor" by polymeric repetitive sequences, a soluble or fixed monomer cannot activate but can inhibit activation, and MAP binding initiates increased expression of a putative NCC receptor protein.

Amino Acid Sequence↗

PubMed1996

Mapping of the epitope recognized by non-specific cytotoxic cells: determination of the fine specificity using synthetic peptides.

NCC recognize a conserved target cell antigen (NKTag) expressed on protozoan parasites and on transformed tumour cells. In the present study, synthetic peptides corresponding to N-terminal, C-terminal and internal NKTag(deduced) amino acid sequences were tested for binding and inhibition of NCC lysis of sensitive target cells. A 20-mer peptide equivalent to amino acids (aa) nos. 55-74 specifically inhibited NCC lysis of human EBV transformed target cells (IM-9). Inhibitory effects were nonreversible and concentration dependent, and 30 min pre-incubation produced optimum inhibition. The inhibitory 20-mer peptide was truncated into 17, 14, 10, 9 and 6-mer peptides and tested for inhibition of cytotoxicity. All produced almost complete inhibition except the 6-mer which had no activity. The NKTag sequence required for NCC binding (minimally) consisted of seven amino acids [aa nos 68-74 (ARG-ASN-LEU-THR-PHE-ILE-LEU-)]. The specificity of inhibition and the distribution of target cells expressing NKTag was determined. A 14-mer peptide composed of aa nos 61-74 inhibited lysis of HL-60, IM-9, DAUDI, YAC-1, U937 and NC-37 target cells. Flanking peptides (aa nos 35-54 and 75-94) were negative. Biotinylated aa nos 61-74 bound to NCC effector cells. The recognition requirements for aa sequence versus aa content were determined. Randomization of the aa in the cognate 9-mer obliterated the inhibitory effects. The 17-mer (cognate) synthetic peptide inhibited conjugate formation between NCC and IM-9 targets. These data demonstrate that NCC recognize a conserved antigen determinant on susceptible target cells consisting of a minimum of 7-9 amino acids in the N-terminal region of NKTag.

Amino Acid Sequence↗

PubMed1995

Monoclonal antibody binding to a receptor on nonspecific cytotoxic cells (NCC) increases the expression of proto-oncogene kinases and protein kinase C.

Teleost nonspecific cytotoxic cells (NCC) initiate various cell triggering responses following receptor-target cell interactions. A putative receptor protein on NCC may alternatively initiate signalling processes following crosslinkage by homologous anti-receptor mab 5C6. In the present study, we demonstrated that binding to this receptor by mab 5C6 produced increased levels of expression of cytoplasmic src family proto-oncogene kinases lck, fyn and src. The phosphorylated isoforms of each kinase were approximately the same molecular weight (p60). Unlike their mammalian T-cell and natural killer (NK) cell counterparts, NCC p56lck did not autophosphorylate on tyrosine residues. This was determined by a lack of Western blot reactivity of teleost p56lck with anti-phosphotyrosine specific antibodies PT-66 or 4G10. Additional evidence for this lack of tyrosine phosphorylation was shown by experiments treating mab 5C6 activated NCC with sodium orthovanadate. This protein tyrosine phosphatase inhibitor did not affect levels of p56lck autophosphorylation. Mab 5C6 activated NCC were also examined to determine if levels of protein kinase C (PKC) expression were affected during triggering responses. Maximum increased PKC levels occurred 5-10 min following binding. The NCC receptor-activated PKC consisted of a 60,000 M(r) isoform and a 30,000 M(r) homologue equivalent to the mammalian PKC catalytic subunit. Not all kinases examined, however, were affected by mab 5C6 binding. Levels of expression of c-myc and cdc2p34 did not change following NCC activation. This is the first study of NK-like cells in cold-blooded vertebrates regarding the expression of these vital intermediary transducing kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Activity-wheel running attenuates suppression of natural killer cell activity after footshock.

We studied whether voluntary running in an activity wheel moderates splenic natural killer (NK) cell cytotoxicity after footshock. Young (50-day) male Fischer 344 rats were randomly assigned to 1) sedentary (n = 16) or 2) activity-wheel (n = 16) groups that each received controllable or uncontrollable footshock on 2 consecutive days or 3) a sedentary home-cage control group (n = 8). Spleens and trunk blood were collected 30 min after the second footshock session. Cytotoxicity was determined by a standard 4-h 51Cr release assay. Percentages of OX6+ (B), OX8+ [T suppressor/cytotoxic (Ts/c)], W3/25+ (T helper), Thy-1.1 (Pan T cell marker), and 5C6+ (NK) cells were determined by flow cytometry. Plasma adrenocorticotropic hormone, corticosterone, and prolactin concentrations were measured by radioimmunoassay as modulators of NK activity. Percentage of specific lysis after footshock was approximately 52% of control values for sedentary animals compared with approximately 96% of control values for activity-wheel animals. The groups did not differ in percentages of NK or Ts/c cells. We conclude that voluntary activity-wheel running protects against the suppression of splenic NK activity induced by footshock. This protective effect of wheel running is not explained by an elevation in baseline NK activity; increased percentages of splenic NK or Ts/c cells; or plasma levels of adrenocorticotropic hormone, corticosterone, and prolactin.

Adrenocorticotropic Hormone↗

PubMed1994

Pathways of signaling in nonspecific cytotoxic cells: effects of protein kinase and phosphatase inhibitors and evidence for membrane tyrosine phosphorylation.

The present study was done to examine the role of protein kinases (PK) and phosphatases in nonspecific cytotoxic cell (NCC) activation processes. Treatment of NCC with 30 or 60 microM H-7 prior to adding IM-9 target cells significantly inhibited cytotoxicity. A similar concentration and time-dependent inhibition response was observed following treatment with H-8. The calcium and cyclic nucleotide antagonist HA1004, however, produced a significant increase in cytotoxicity at 30 and 60 microM concentrations. Treatment with genistein produced almost 100% inhibition of NCC lysis of IM-9 targets at a 142 micrograms/ml concentration. Studies were also carried out to determine the effects of these cytotoxicity modulators on stimulus-induced responses. In all cases where the costimulus consisted of the calcium ionophore A23187, cytotoxicity was markedly increased. In experiments using subsaturating concentrations of mab 5C6 as the costimulus, genistein completely reversed (inhibited) the modulating effects of this mab. Additional evidence for protein phosphorylation was obtained by immunoprecipitation and Western blot analysis (using antiphosphotyrosine mab 4G10) of membranes from mab 5C6-stimulated NCC. The role of protein phosphorylation on cytotoxic activity was further analyzed by the use of phosphatase inhibitors. Preincubation of NCC with either sodium orthovanadate or sodium fluoride significantly increased cytotoxicity. Lithium chloride had little or no effect on the killing of IM-9 target cells. These data indicate that multiple second messenger pathways participate in NCC lytic responses, the most crucial of which appears to facilitate phosphorylation of tyrosine residues on membrane proteins.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1994

Target-cell sensitivity to natural killer-cell lysis is determined by the expression of a novel antigen in conjunction with major histocompatibility complex class-I molecules.

Recently, a panel of monoclonal antibodies (MoAbs) was developed that identified a novel tumour-cell antigen conserved across species (mouse, rat and man). Fluorescence-activated cell sorter (FACS) analysis demonstrated that this antigen was expressed at highest levels on human tumour cell lines sensitive to natural killer (NK)-cell lysis. These MoAbs inhibited NK cell lysis of K562 target cells (by up to 90%), as well as a variety of other NK-sensitive target cells. Biochemical analyses revealed that the MoAbs reacted with a polypeptide of 42 kDaltons, distinct from other known cell surface antigens. Now the expression of this antigen has been analysed further with a panel of 24 tumour cell lines to determine its role in NK cell function. The expression of target cell major histocompatibility complex (MHC) molecules in conjunction with sensitivity to NK cell lysis was examined also. For each of the 24 cell lines it was found that the level of expression of the novel target cell antigen determined the sensitivity of the cell line to NK cell lysis. However, the level of MHC antigen expression could modulate target cell sensitivity to NK cell lysis, in that high levels of MHC class-I molecule expression resulted in a target cell that was insensitive to NK cell lysis regardless of the level of expression of the novel antigen. Thus, for most transformed cell lines, sensitivity to NK cell lysis appeared to be determined by the expression and levels of the novel antigen in association with MHC class-I molecules.

Adolescent↗

PubMed1994

Partial amino acid sequence of a novel protozoan parasite antigen that inhibits non-specific cytotoxic cell activity.

Monoclonal antibody (MoAb) 18C2, prepared against a human EBV transformed lymphoblastic cell line (NC-37) is specific for a target cell ligand recognized by fish NCC and by mammalian NK cells. MoAb 18C2 inhibits the lysis of a variety of transformed murine and human cells (e.g. NC-37, YAC-1, K562, etc.). This MoAb also recognizes a determinant on the fish protozoan parasite Tetrahymena pyriformis. In the present study, we used MoAb 18C2 to identify a target antigen in detergent lysates of T. pyriformis. MoAb 18C2 recognized a 46-50 kDa target antigen (NKTag) by Western blot analysis of both crude and ammonium sulphate (AS) fractionated (25-40% saturation) T. pyriformis lysates. AS fractionated or purified soluble NKTag inhibited NCC mediated lysis of IM-9 target cells in a dose dependent fashion. AS fractionated NKTag also inhibited NCC lysis of a variety of human and murine transformed targets (e.g. HL-60, MOLT-4, DAUDI, NC-37, U-937, YAC-1, EL-4). Inhibition was specific for NCC and inhibition could be removed by adsorption of AS fractionated NKTag with MoAb 18C2 hybridoma cells. NKTag was prepared for amino acid sequencing by preparative SDS PAGE of whole cell detergent (CHAPS) lysate followed by Western transfer to nitrocellulose. The MoAb 18C2 recognized NKTag was excised and submitted for microsequence analysis. Direct N-terminal analysis yielded a 12 residue sequence. Additional sequences, obtained from in situ trypsin digests of the NKTag on nitrocellulose yielded four additional peptides of 10, 13, 16 and 21 residues. None of the sequences examined had significant homology to known sequences (Swiss-Prot protein sequence database). These data indicate that MoAb 18C2 recognized a novel protein on T. pyriformis which may be involved in target cell recognition/lysis by NCC. Further, these data extend our previous observation that a common target determinant exists between higher and lower eukaryotic cells, and its expression may provide an explanation for the susceptibility of both protozoan parasites and transformed tumour cells to NK/NCC lysis.

Amino Acid Sequence↗

PubMed1993

Nonspecific cytotoxic cells in fish: antigenic cross-reactivity of a function associated molecule with the intermediate filament vimentin.

Nonspecific cytotoxic cells (NCC) in fish may lyse tumor cells and protozoan parasites in a similar fashion as their mammalian NK counterparts. Previous studies have shown that recognition and binding by NCC may be mediated by a receptor. In the present study the putative receptor or function-associated molecule (FAM) on NCC was shown to contain antigenic cross-reactivity with the mammalian intermediate filament vimentin. This was accomplished by comparing the specificity of anti-FAM monoclonal antibody 5C6 with commercially available anti-vimentin mabs V9 and 13.2 and with a polyclonal anti-vimentin antiserum. Tissue distribution studies indicated that the anti-vimentin antibodies bound to significantly fewer (< 50%) anterior kidney, spleen, kidney, and brain cells than mab 5C6. These mabs, however, produced inhibition of NCC lysis of IM-9 target cells. Conditions of mab incubation (with NCC) which were previously shown to modulate NCC cytotoxicity by 5C6 also produced large increases in NCC lysis of IM-9 targets in the presence of mabs V9 and 13.2. The determinant(s) recognized by these antibodies is present on the cell membrane. Microscopic examination of nonpermeabilized or acetone-treated and antibody-stained cells demonstrated that acetone permeabilization produced a 28-32% increase in staining (due to intracellular binding) compared to the staining of viable cells. Biochemical analysis of the cross-reactive antigens was done using analytical 2D SDS-PAGE and Western blot experiments. Mab 5C6 detected a 32- to 35-kDa protein (pI 6.3-6.5) in NCC extracts. Anti-vimentin mab 13.2 recognized a 57-kDa vimentin protein which had a relatively broad pI range (pH 6.3-6.7). Mab 13.2 also cross-reacted (in the same Western blot) with the NCC receptor protein (pI = pH 6.3-6.5). These data indicate that the FAM on NCC/NK cells contain vimentin-like antigenic determinants which may function in target cell binding.

Animals↗

PubMed1993

Natural killer (NK) cells in domestic animals: phenotype, target cell specificity and cytokine regulation.

A comprehensive review of NK cells in animals of veterinary medical importance has not been previously published. However, these cells have a high level of immunological/medical relevance due to their role in tumour cell destruction and B-cell regulation, as well as their inhibitory activities against various parasites and bacteria. In the present review, NK cells from agriculturally important animals are characterized. Cell phenotype descriptions have shown that for each species, unique (i.e. non-cross-reactive with anti-human CD antibodies) and different monoclonals are required to identify NK cells. These cells lyse certain tumour and virus transformed target cells and, as might be expected from the diverse species compared in this review, analysis of the tissue distribution of NK cells gives highly varied results. NK cell morphology differs in these species from small agranular to large granular lymphocytes. The final area considered relates to studies on the effects of cytokines on NK function and to research identifying which cytokines (if any) are produced by NK cells during activation responses. The largest quantity of available data concerns functional and descriptive studies in these animals. However, it is appropriate to consider this research as a starting point on which to build comparative and molecular studies of the roles of NK cells in immunosurveillance and immunoregulation.

Animals↗

PubMed1993

Expression of an evolutionarily conserved function associated molecule on sheep, horse and cattle natural killer cells.

Natural killer (NK) cells are large granular lymphocytes that lyse a wide variety of transformed and virally-infected target cells without prior exposure to antigen, and without restriction by major histocompatibility complex antigens. Although NK cells have been identified in a variety of mammalian species, how NK cells recognize antigen and trigger lysis is unknown. Recently, monoclonal antibodies made against NK-like cells from teleost fish were shown to react with NK cells from humans and rats, and to inhibit their cytolytic activity. The role of this apparently evolutionarily conserved function-associated molecule (FAM) has been further investigated utilizing a variety of domesticated farm animal species. It was observed that the anti-FAM mAb reacted specifically with peripheral blood lymphocytes isolated from sheep, horses and cattle. Further, the anti-FAM mAb inhibited NK cell lytic activity in each of these species. Finally, the anti-FAM mAb was found to inhibit conjugate formation between NK and target cells, implying that the FAM was involved in antigen recognition by NK cells in each of these species. In conclusion, it appears that NK cell function is mediated by an evolutionarily conserved FAM in a wide variety of species.

Animals↗

PubMed1993

A novel target cell antigen involved in the NK-like lytic activity of antigen-specific cytotoxic T lymphocytes.

Monoclonal antibodies (mAbs) have been derived which identify a target cell antigen involved in human natural killer (NK) cell lysis. The effects of the mAbs on the NK-like cytotoxic activity exhibited by different populations of human cytolytic T cells were examined. The anti-target cell mAbs 1E7 and 18C2 inhibited the lysis of K562 target cells by endogenous NK cells, antigen-specific cytotoxic T lymphocytes (CTL) with NK-like activity, and non-major histocompatibility complex (MHC)-restricted T cells. Cytolysis of K562 target cells was not affected by treatment of the target cells with the anti-class I HLA mAb W6/32. Further, the anti-target cell mAbs had no effect on antigen-specific lysis by the CTL. The mAb W6/32, however, inhibited the antigen-specific killing. Experiments at the single-cell level revealed that the anti-target cell mAbs inhibited the formation of conjugates between the effector cells and K562 tumor cells but had no effect on CTL binding to the antigen-specific target cells. Thus, antigen-specific CTL exhibiting NK-like lytic function appeared to recognize a novel target cell antigen that is distinct from typical MHC antigens and is identical to the target-cell antigen recognized by true NK cells.

Adolescent↗

PubMed1992

Detection of function-associated molecules on rat NK cells and their role in target cell lysis.

Anti-effector cell mAb 5C6.10.4 (5C6) inhibits cytotoxic activity of fish nonspecific cytotoxic cells (NCC). We now show that 5C6 also inhibits mammalian NK cell activity using fresh and cultured (CRC) leukemic rat NK cells. The inhibitory activity of 5C6 was caused by blocking of conjugate formation between NK cells and YAC-1 targets. Binding studies done by flow cytometry (FCM) showed that mAb 5C6 specifically bound to 8% of unfractionated rat spleen cells. Enrichment by nylon-wool fractionation produced 27.2% specific binding, along with a 3.4-fold enrichment in cytotoxic activity. Tissue distribution studies revealed that the highest number of cells recognized by mAb 5C6 were found in NWNA spleen cells (28.7%), followed by liver (18.9%) and peripheral blood (13.9). Two-color FCM showed that although all 3.2.3 mAb-positive cells were also stained with mAb 5C6, a small percentage of 3.2.3. negative noncytotoxic NWNA spleen T cells were 5C6 positive. Redirected lysis experiments demonstrated that anti-effector mAb-producing myeloma cells could be killed by CRC and NWNA spleen cells. In addition, mAb 5C6 produced specific inhibition of redirected lysis of each myeloma target. Experiments were also conducted to determine the signaling capability of the FAM complex. Binding of the anti-FAM mAbs to NWNA rat spleen cells caused a rapid increase in cytosolic free calcium of approximately 472 nM. Western blot analysis of CRC cell lysates showed that the molecules recognized by anti-FAM mAbs have molecular weights of 38 and 42 kDa. These studies indicate that the anti-effector mAbs recognize a functionally relevant molecule on rat NK cells that is involved in the first steps of cytolysis, i.e., antigen recognition, and which also triggers the activation of signal-transducing events in these cells.

Animals↗

PubMed1992

A function-associated molecule on rat natural killer cells identified by anti-idiotypic monoclonal antibodies.

Monoclonal antibodies were generated against idiotopes on an NK target antigen-specific IgM monoclonal antibody (mab). This mab (18C2) was originally produced against (NC-37) human EBV-transformed B cells. The 18C2 mab inhibits natural killer cell lysis of NC-37 and other target cells by preventing conjugate formation. Anti-18C2(id) mabs were tested for binding to effector cells and screened by ELISA, flow cytometry, and by inhibition of NK cytotoxicity. Two of the anti-18C2(id) (anti-id) mabs (12H1.C5 and 6D9.B11) were chosen for further study. The idiotypic specificity of these anti-id mabs was confirmed by testing their binding to 18C2 hybridoma cells in the presence of homologous and heterologous "cold" inhibitor mabs. Experiments were also conducted to determine the functional properties of these mabs. Anti-18C2(id) mab 12H1.C5 inhibited the cytotoxic activity of rat splenic NK (nylon wool nonadherent cells, NWNA) and rat ALAK cells. Flow cytometric (FCM) analysis of the binding of the anti-18C2(id) mabs demonstrated that mab 12H1.C5 bound 75.43% rat NWNA spleen cells, 43.74% rat ALAK cells, and 74.33% rat CRC- cells. Anti-id mab 6D9.B11 bound 45.20% NWNA cells, 70.45% rat ALAK cells, and 55.86% CRC- cells. Two-color FCM analysis demonstrated that the anti-id mabs not only bound to the same molecule on NK cells, but also these mabs bound to the same molecule as 5C6, an anti-NK cell mab. Biochemical analysis of the antigen recognized by mab 12H1.C5 was determined by Western blotting. The determinant on NWNA cells recognized by mab 12H1.C5 had an M(r) of 40 kDa and appeared to be identical to that recognized by mab 5C6. The same experiment using a transformed rat RNK-16 (CRC-) cell extract and Western blot analysis, demonstrated an M(r) of 42 and 48 kDa in the presence of mabs 5C6 and 12H1.C5. Monoclonal antibody 5C6 was previously shown to recognize a vimentin-like function-associated molecule on NK cell membranes. The anti-id mabs were also shown to have cross-reactivity with the intermediate filament vimentin as determined by Western blot analysis.

Animals↗

PubMed1992

The natural killer cell-like lytic activity expressed by cytolytic T lymphocytes is associated with the expression of a novel function-associated molecule.

Experiments were performed to analyse the natural killer cell (NK)-like cytotoxicity frequently expressed by human antigen-specific cytolytic T lymphocytes (CTL). To this end, several monoclonal antibodies (MoAbs) previously shown to identify a novel function-associated molecule (FAM) involved in human NK function were utilized. Flow cytometry revealed that these MoAbs reacted with the majority of human NK, but only with a subpopulation of CTL isolated from primary mixed lymphocyte cultures. Preincubation of CTL with the MoAbs inhibited the NK-like lysis of K562 targets. Experiments with anti-CD3 MoAb demonstrated that neither the NK-like cytotoxicity of CTL nor the lytic activity of NK were mediated by the CD3 complex. Expression of the novel FAM was found to develop in T-cell cultures at the time that NK-like cytotoxicity was observed. Repeated in vitro antigenic stimulation of CTL was shown to result in loss of NK-like cytotoxicity, as well as loss of the FAM on the CTL surface. Thus, NK-like cytotoxicity displayed by antigen-specific CTL appears to be mediated by a novel FAM that is identical to that structure found on NK.

Adolescent↗

PubMed1992

Effect of age on activation of porcine intestinal guanylate cyclase and binding of Escherichia coli heat-stable enterotoxin (STa) to porcine intestinal cells and brush border membranes.

Development of age-dependent resistance to enterotoxigenic Escherichia coli was studied, using isolated enterocytes and brush border membranes (BBM) from 7-day-old and 7-week-old pigs. Binding of 125I-labeled heat-stable (125I-STa) enterotoxin to enterocytes and BBM was specific, temperature- and time-dependent, saturable, and partially reversible. Scatchard analysis revealed a single class of receptors. Mean +/- SD avidity of binding (apparent affinity constant, Ka) of 125I-STa to enterocytes from 7-day-old and 7-week-old pigs was 2.14 +/- 0.29 x 10(8) and 2.72 +/- 0.25 x 10(8) L/mol, respectively. Numbers of STa receptors were calculated to be 64,903 +/- 2,900/enterocyte for 7-day-old pigs and 53,029 +/- 3,117/enterocyte for 7-week-old pigs. Numbers of STa receptors expressed per milligram of BBM protein from 7-day-old pigs were 2.66 x 10(11), compared with 2.29 x 10(11) for BBM from 7-week-old pigs. By 5 minutes after addition of STa to reaction mixtures, intracellular cyclic guanosine monophosphate concentration increased 13.9-fold in enterocytes from 7-day-old pigs and 8.7-fold in enterocytes from 7-week-old pigs. The particulate guanylate cyclase activity associated with BBM from 7-week-old pigs was slightly more sensitive to low amounts of STa, compared with BBM from 7-day-old pigs; however, differences were not observed at intermediate and high amounts. These data indicate that lack of a secretory response to STa by older pigs is not attributable either to decreased numbers of STa receptors or to decreased signal response between the STa receptor and membrane-bound guanylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1991

Identification of an evolutionarily conserved, function-associated molecule on human natural killer cells.

Nonspecific cytotoxic cells (NCC) in teleost fish are analogous to human natural killer (NK) cells and spontaneously lyse a variety of transformed human cell lines sensitive to human NK cell lysis. Monoclonal antibodies (mAbs) made against these NK-like effector cells inhibited their lytic activity. These anti-NCC mAbs were examined with human NK cells for their effects. It was observed that these mAbs bound specifically to a small percentage of peripheral blood lymphocytes (5-15%) and to the majority (greater than 85%) of CD3- NK cells. The mAbs inhibited human NK cell lysis against a variety of transformed cell lines. Single-cell assays showed that the mAbs exerted their effects through inhibition of conjugate formation (recognition). However, the mAbs did not inhibit NK cell-mediated antibody-dependent cell-mediated cytotoxicity. Biochemical analysis of the NK cell molecule revealed that a dimeric structure was involved (distinct from antigen receptors on T cells). Thus, this molecule appears to be a candidate NK cell antigen receptor.

Antibodies, Monoclonal↗