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N W Miller

Publications and source records attributed to N W Miller.

At least 37 records · Page 2Linked to original sources
PubMed1998

Evolutionary variation of immunoglobulin mu heavy chain RNA processing pathways: origins, effects, and implications.

Immunoglobulins (Ig) can occur in two physical forms, soluble (secreted) and membrane bound. The soluble form is secreted from B cells, and is present in the blood and other fluids where it plays a role as an immune effector molecule. The membrane-bound form of the Ig molecule is inserted into the B-cell membrane, where it serves as a receptor for antigen. The function of the membrane-bound Ig as a receptor for antigen requires additional accessory molecules, the membrane Ig plus accessory molecules are referred to, collectively, as the B-cell receptor (BCR) complex. The secreted and membrane-bound forms of an Ig result from alternative patterns of RNA processing of the primary transcript from the heavy chain gene. IgM is the only class of Ig known to be conserved in all vertebrate species (perhaps exclusive of the agnathan fish). While the structure of the IgM heavy (mu) chain gene has been highly conserved in vertebrate evolution, the patterns of alternative RNA processing of the mu transcript show surprising diversity. In particular, the bony fish (teleosts) produce membrane mu-chain message by a splicing pathway that is quite different from that seen in other vertebrates; it results in the production of membrane IgM that lacks the C mu 4 domain. How this unusual RNA splicing pattern could have evolved and its implications for the function of the BCR in the bony fishes are considered here.

Animals↗
PubMed1997

Catfish thrombocytes express an integrin-like CD41/CD61 complex.

A thrombocyte-specific antigen was identified in two closely related catfish, Ictalurus punctatus and Ictalurus furcatus, by monoclonal antibodies 4-20 and 7-2. The antibodies immunoprecipitate two noncovalently associated glycoprotein chains of Mr 180,000 and Mr 95,000. Under reducing conditions the Mr 180,000 chain is resolved into Mr 150,000 and 32,000 subcomponents. Analysis of N-terminal amino acid sequences indicates homology of the Mr 95,000 chain with the beta3 integrin subunit and homology of the Mr 150,000 chain with the alphaIIb integrin subunit. These antibodies induce catfish thrombocyte aggregation and alteration of cell shape. The data indicate conservation of the megakaryocyte/platelet-restricted CD41/CD61 complex in bony fish.

Amino Acid Sequence↗
PubMed1997

Cytotoxic activity generated from channel catfish peripheral blood leukocytes in mixed leukocyte cultures.

In previous work, lysis of allotargets was routinely observed with PBL from nonimmune channel catfish. In the work reported here, greatly increased (approximately 100-fold) cytotoxic responses were generated by stimulation of channel catfish PBL with irradiated cells of allogeneic cloned B cell lines in mixed leukocyte cultures (MLC). This increased cytotoxicity did not appear to be simply a consequence of cell proliferation since stimulation of catfish PBL proliferative responses with polyclonal mitogens did not result in increased lysis. Somewhat surprisingly, the MLC-generated cytotoxicity did not exhibit allospecificity; i.e., allogeneic targets from other fish were as susceptible to lysis as were the cells used as stimulators. This apparent lack of allospecificity in MLC-generated cytotoxicity was confirmed by "cold" target inhibition assays. However, autologous targets were not killed, clearly demonstrating that MLC-generated effectors could distinguish "self" from "nonself" at the level of lysis/recognition. Although their origin is unresolved, the MLC-generated effectors may be a source of highly enriched fish cytotoxic cells and thus facilitate directly addressing questions pertaining to the evolution of such cells.

Animals↗
PubMed1997

A novel chimeric Ig heavy chain from a teleost fish shares similarities to IgD.

IgD is considered to be a recently evolved Ig, being previously found only in primates and rodents. Here we describe, from a teleost fish (the channel catfish, Ictalurus punctatus), a novel complex chimeric Ig heavy chain, homologous, in part, to the heavy chain (delta) of IgD. In addition to alternative secretory or membrane-associated C termini, this chimeric molecule contains a rearranged variable domain, the first constant domain of mu, and seven constant domains encoded by a delta gene homolog. Identification of the catfish gene as delta is based on the following properties: sequence relatedness to mammalian delta; a location within the IgH locus that is immediately downstream of the mu gene; separate terminal exons for the secretory and membrane forms; coexpression with the complete mu chain in some but not all B cells. These results (i) suggest that IgD is an ancient immunoglobulin that was present in vertebrates ancestral to both the mammals and the ray-finned fishes, and (ii) raise the possibility that this Ig isotype may have served an as yet unidentified important function early in the evolution of the immune system.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of a heat shock protein 70 family member in channel catfish (Ictalurus punctatus).

We have determined the cDNA sequence of a member of the channel catfish heat shock protein 70 (CF Hsp70) family. This protein presumably functions as a molecular chaperone, as is characteristic of this family in other species. Channel catfish peripheral blood leukocytes exhibit a classical heat shock response, in that heat shock (37 degrees C) induces the expression of heat shock genes that are quiescent at normal temperatures (23 degrees C). It was observed that pre-existing synthesis of certain other molecules was suppressed (as evidenced by decreases in actin RNA upon heat shock). Similar trends were observed in mRNA expression for CF Hsp70 in two catfish non-leukocyte cell lines, channel catfish ovary and F59. However, three leukocyte cell lines constitutively expressed high levels of CF Hsp70 mRNA at optimal culture temperature (27 degrees C), whereas heat shock (37 degrees C) elicited only a modest induction of CF Hsp70 expression. Furthermore, continued investigation is warranted to determine whether the apparent upregulation of CF Hsp70 mRNA expression in the catfish long-term leukocyte cell lines is involved in the seemingly immortal phenotype of these cells.

Amino Acid Sequence↗
PubMed1996

Fish immunology: the utility of immortalized lymphoid cells--a mini review.

Long term cell lines can be readily established at high frequency with PBLs from normal channel catfish. Depending upon the mode of stimulation, morphologically and functionally distinct catfish lymphoid cell lines resembling B cells, T cells and monocytes have been developed. These fish cell lines appear unique from their putative mammalian counterparts in that they are immortalized without the need for exogenous factors or overt attempts at transformation.

Animals↗
PubMed1994

An Ig heavy chain enhancer of the channel catfish Ictalurus punctatus: evolutionary conservation of function but not structure.

The teleost fishes are among the earliest evolutionary lineages to have an Ig heavy chain (IgH) locus whose organization approximates that of mammals. To understand transcriptional control of the IgH locus in a teleost fish and to gain insight into the evolution of the control elements, the enhancer activity in the IgH locus of the channel catfish, Ictalurus punctatus, was investigated. Segments of the locus extending from upstream of the proximal JH gene to 2.5 kb downstream of the second transmembrane (TM2) exon of the mu gene were tested in transient transfection expression assays in murine myeloma and T cell lines, and in catfish B lymphoblastoid, monocyte-like, and putative T cell lines. In marked contrast to mammals, no enhancer activity was observed in the catfish JH to C mu intron, but strong enhancer activity (approaching that of the murine IgH intronic enhancer) was identified in a 1.8-kb segment that included the TM2 exon. This catfish enhancer was active in a B lineage-specific manner in both catfish and murine cells. It was not localized in a small core region, but appeared to contain multiple, dispersed cooperative elements rich in octamer- and mu E5-related motifs. Although the catfish IgH enhancer shares functional characteristics with the mammalian IgH intronic and 3' enhancers, its unusual organization does not permit any obvious inferences concerning evolutionary relationships between the catfish enhancer and any one of the murine IgH enhancers.

Animals↗
PubMed1994

Development and characterization of channel catfish long term B cell lines.

The establishment of channel catfish long term cloned B cell lines, the first such cell lines from ectothermic vertebrates, is described. These diploid cell lines were developed by in vitro LPS stimulation of B cells from normal channel catfish peripheral blood in the absence of overt attempts to transform or immortalize the cells. The resultant cell lines were cloned and maintained continuously in vitro for more than 12 mo without restimulation, feeder cells, or exogenous factors. Southern blot analyses of the parental cell lines revealed multiple mu-chain gene rearrangements, suggesting a polyclonal origin for the cell lines. Additional evidence for polyclonal development was provided by the demonstration that the parental cell lines transcribed mRNA for all of the six known channel catfish VH gene families. The characterization of several clonal cell lines revealed mRNA expression for both the secreted and membrane forms of the catfish mu-chain; however, the cloned cell lines each expressed only a single VH gene and analysis of the Ig H chain locus was consistent with allelic exclusion having occurred in these cells. Flow cytometry demonstrated that the cloned and uncloned cell lines produced both cytoplasmic and cell surface IgM. This IgM contained only one of the two L chain isotypes of the channel catfish, suggesting preferential L chain usage. Although these cells did not appear morphologically to be plasma cells, they secreted moderate levels of IgM in culture. These cell lines have considerable potential for addressing questions concerning the evolution of B cell function.

Animals↗
PubMed1993

Productive infection of continuous lines of channel catfish leukocytes by channel catfish virus.

Channel catfish virus (CCV) undergoes extensive replication in fingerling catfish and establishes latent infection in survivors. Although the site of viral latency in carriers is unknown, a variety of tissues, including leukocytes, have been implicated. To explore the interaction of CCV and leukocytes further, we examined in vitro infection of continuous lines of cloned catfish B cells and non-cloned lines containing predominantly macrophages and putative T-cells. Our results indicate that all three leukocyte subpopulations were productively infected with CCV, but that the kinetics of infection and the final yields differed markedly. These findings shed light on CCV-leukocyte interaction and suggest that CCV-infected autologous macrophage or T-cell lines might be suitable targets for assays of catfish cytotoxic T cell activity.

Animals↗
PubMed1993

Expression of a mouse-channel catfish chimeric IgM molecule in a mouse myeloma cell.

Fusion genes encoding a murine VH domain and the constant region domains of the mu chain from the channel catfish, Ictalurus punctatus, were stably expressed in the lambda light chain producing mouse myeloma cell line J558L. Although the pathways of pre-mRNA processing for expression of membrane (micron and secreted (microsecond) forms of the mu chain differ between mammals and teleosts, mRNAs encoding both catfish micron and microsecond were correctly expressed in the mouse myeloma cells. The mouse-channel catfish chimeric mu chain polypeptide was able to associate covalently with the mouse lambda light chain and assemble, intracellularly, into polymers of covalent structure (microL)2-8 which resembled those seen with native catfish IgM. In contrast to native catfish IgM, the mouse-catfish chimeric IgM showed the property of binding strongly to protein A of Staphylococcus aureus. The mouse-channel catfish chimeric IgM was core-glycosylated, but did not contain terminal sialic acid. Secretion rates for the chimeric IgM were low, and the possibility could not be excluded that extracellular chimeric IgM was released from dead or dying cells. The reason(s) for the intracellular retention of the chimeric IgM (probably in the endoplasmic reticulum) are not known, but those mechanisms involving retention via cysteine residues were excluded.

Animals↗
PubMed1992

Alternate splicing pathways of the immunoglobulin heavy chain transcript of a teleost fish, Ictalurus punctatus.

Previously we sequenced a partial cDNA clone encoding the 3' region of the message for the membrane receptor form of the heavy (mu) chain of the channel catfish which indicated that the first transmembrane (TM1) exon is spliced directly to the C mu 3 exon and not into a cryptic site within the CH4 exon, as occurs in other vertebrates. Studies utilizing polymerase chain reaction analysis of mRNA and further analysis of cDNA clones now confirm that the only detectable splicing pattern used in micron production by the channel catfish utilizes this C mu 3----TM1 pathway of pre-mRNA splicing.

Animals↗
PubMed1992

Cellular pathway(s) of antigen processing and presentation in fish APC: endosomal involvement and cell-free antigen presentation.

Studies were conducted to address further the role(s) of antigen processing and presentation in the induction of immune responses in a phylogenetically lower vertebrate, specifically a teleost, the channel catfish. In particular, studies were aimed at determining the subcellular compartments involved in antigen degradation by channel catfish antigen-presenting cells (APC) as well as ascertaining the reexpression of immunogenic peptides on the surfaces of APC. The results showed that exogenous protein antigens were actively endocytosed by APC as detected by flow cytometry. Use of radiolabeled antigen and subcellular fractionation protocols also showed that antigen localized in endosomes/lysosomes. Furthermore, there was an apparent redistribution of antigen between these organelles and the plasma membrane during the course of antigen pulsing. Functional assays for the induction of in vitro antigen-specific proliferation of immune catfish peripheral blood leukocytes (PBL) showed that membrane preparations from antigen-pulsed autologous APC were highly stimulatory. The magnitude of responses elicited with such membrane preparations was very similar to that of PBL cultures stimulated with native antigen-pulsed and fixed intact APC or prefixed intact APC incubated with a peptide fragment of the nominal antigen. Current data further corroborate our previous findings that steps akin to antigen processing and presentation are clearly important in the induction of immune responses in lower vertebrates like fish, in a manner similar to that seen in mammalian systems. Consequently, it would appear that many immune functions among the diverse taxa of vertebrates are remarkably conserved.

Animals↗
PubMed1992

Doctors must review referral and business relationships.

Ten safe harbors describe referral relationships and other business arrangements exempt from prosecution under federal anti-kickback law. As the statute's prohibitions are broad and the safe harbors narrow, physicians should review--carefully--their practice relationships.

Commerce↗
PubMed1991

Spontaneous development of functionally active long-term monocytelike cell lines from channel catfish.

During the course of studies involving the in vitro manipulation of channel catfish peripheral blood leukocytes, spontaneous proliferation was observed with unexpectedly high frequency. Propagation of these spontaneously proliferating cells has resulted in the development of long-term (greater than 11 mo.) cell lines which stain positively for nonspecific esterase and peroxidase, are phagocytic for latex beads, and morphologically resemble mammalian monocytes or macrophages. These long-term cell lines also exhibit two important additional functional features. First, induction with lipopolysaccharide results in the secretion of relatively high levels of catfish high and low molecular weight species of interleukin-1 active on channel catfish and mouse T cells, respectively. Second, these cell lines are efficient antigen-presenting cells to autologous peripheral blood leukocytes for antigen specific in vitro proliferative and antibody responses. This antigen-presenting function is blocked by inhibitors known to prevent antigen processing and presentation by mammalian monocytes. Allogeneic mixtures of cell line (used as antigen-presenting cells) and responding peripheral blood leukocytes, however, resulted in strong mixed leukocyte reaction but not in specific antibody responses. The availability of such cell lines should facilitate further studies on accessory cell functions in fish immune responses.

Animals↗
PubMed1991

Correlation between low-temperature immunosuppression and the absence of unsaturated fatty acid synthesis in murine T cells.

1. Studies were performed to determine if the inability of murine T cells to provide primary helper function at low temperature (27 degrees C) could be correlated with their inability to synthesize unsaturated fatty acids (UFAs). 2. In the absence of exogenous oleic acid (18:1), splenocytes responded to a T-dependent (TD) Ag (trinitrophenyl-keyhole limpet hemocyanin, TNP-KLH) at 37 degrees C but not at 27 degrees C. The addition of 150 microM 18:1 almost completely restored plaque-forming cell (PFC) responses to TNP-KLH at 27 degrees C but markedly suppressed PFC responses to the TD Ag at 37 degrees C. 3. During incubation at 27 degrees C, B cells converted 3- to 5-fold more stearic acid (18:0) to 18:1 and showed a greater accumulation of monounsaturated phospholipid molecular species than did T cells. 4. Following incubation in the presence of a rescuing dose of 18:1 (150 microM), both B and T cells accumulated large amounts of dioleoyl PC. 5. It is proposed that the absence of 18:1 synthesis in T cells is responsible for the unique low temperature susceptibility of this lymphocyte population.

Animals↗
PubMed1991

Phylogeny of immune recognition: processing and presentation of structurally defined proteins in channel catfish immune responses.

This work was undertaken to investigate whether or not antigen processing and presentation are important in channel catfish in vitro secondary immune responses elicited with structurally defined proteins, namely, pigeon heart cytochrome C (pCytC), hen egg lysozyme, and horse myoglobin. The use of in vitro antigen-pulsed and fixed B cells or monocytes as antigen presenting cells (APC) resulted in autologous peripheral blood leukocytes (PBL) responding with vigorous proliferation and antibody production in vitro. In addition, several long-term catfish monocyte lines have been found to function as efficient APC with autologous but not allogeneic responders. Subsequent separation of the responding PBL into sIg- (T-cell-enriched) and B (sIg+) cells subsets showed that both underwent proliferative responses to antigen-pulsed and fixed APC. Moreover, allogeneic cells used as APC were found to induce only strong mixed leukocyte reactions without specific in vitro antibody production. Initial attempts at identifying the immunogenic region(s) of the protein antigens for catfish indicated there are two such regions for pCytC, namely, peptides 66-80 and 81-104.

Animals↗
PubMed1991

Phylogeny of immune recognition: role of alloantigens in antigen presentation in channel catfish immune responses.

Previous work from this laboratory demonstrated the importance of antigen processing and presentation in the generation of secondary in vitro immune responses of channel catfish to both simple and complex T-dependence antigens. Further, the usage of functionally active long-term monocyte lines as antigen-presenting cells (APCs) revealed 'putative restriction' of immune responses by peripheral blood leucocytes (PBL) as responders. In addition, these long-term cell lines proved to be effective antigens for the production of specific alloantisera. Studies were therefore conducted to determine the importance of these alloantigens in the immunological recognition of foreign T-dependent antigens in the channel catfish. The addition of homologous, but not heterologous, alloantisera to cultures of antigen-pulsed cell line APCs and autologous immune PBL responders resulted in quantitative decreases in the antigen-specific proliferative responses. Taken together, these results provide evidence that alloantigens [presumably major histocompatibility complex (MHC) or MHC-like molecules] govern antigen presentation and putative restriction of teleost immune responses similar to the situation in mammals.

Animals↗