E. Churchill McKinney

Carcinoembryonic antigen present in human eccrine sweat

Carcinoembryonic antigen is detectable by standard unlabeled peroxidase-antiperoxidase technics in eccrine and apocrine secretory and ductal cells. Carcinoembryonic antigen is also present in the cuticle which lines the duct. In eccrine sweat, carcinoembryonic antigen activities were at least thirty times that in serum, using a radioimmunoassay. Carcinoembryonic antigen may form part of the Schiff-positive cuticle and function in the normal homeostasis of eccrine and apocrine glands.

Proteasome, Transporter Associated with Antigen Processing, and Class I Genes in the Nurse Shark Ginglymostoma cirratum: Evidence for a Stable Class I Region and MHC Haplotype Lineages

Cartilaginous fish (e.g., sharks) are derived from the oldest vertebrate ancestor having an adaptive immune system, and thus are key models for examining MHC evolution. Previously, family studies in two shark species showed that classical class I (UAA) and class II genes are genetically linked. In this study, we show that proteasome genes LMP2 and LMP7, shark-specific LMP7-like, and the TAP1/2 genes are linked to class I/II. Functional LMP7 and LMP7-like genes, as well as multiple LMP2 genes or gene fragments, are found only in some sharks, suggesting that different sets of peptides might be generated depending upon inherited MHC haplotypes. Cosmid clones bearing the MHC-linked classical class I genes were isolated and shown to contain proteasome gene fragments. A non-MHC-linked LMP7 gene also was identified on another cosmid, but only two exons of this gene were detected, closely linked to a class I pseudogene (UAA-NC2); this region probably resulted from a recent duplication and translocation from the functional MHC. Tight linkage of proteasome and class I genes, in comparison with gene organizations of other vertebrates, suggests a primordial MHC organization. Another nonclassical class I gene (UAA-NC1) was detected that is linked neither to MHC nor to UAA-NC2; its high level of sequence similarity to UAA suggests that UAA-NC1 also was recently derived from UAA and translocated from MHC. These data further support the principle of a primordial class I region with few class I genes. Finally, multiple paternities in one family were demonstrated, with potential segregation distortions.

Evolutionarily Conserved TCR Binding Sites, Identification of T Cells in Primary Lymphoid Tissues, and Surprising Trans-Rearrangements in Nurse Shark

Cartilaginous fish are the oldest animals that generate RAG-based Ag receptor diversity. We have analyzed the genes and expressed transcripts of the four TCR chains for the first time in a cartilaginous fish, the nurse shark (Ginglymostoma cirratum). Northern blotting found TCR mRNA expression predominantly in lymphoid and mucosal tissues. Southern blotting suggested translocon-type loci encoding all four chains. Based on diversity of V and J segments, the expressed combinatorial diversity for γ is similar to that of human, α and β may be slightly lower, and δ diversity is the highest of any organism studied to date. Nurse shark TCRδ have long CDR3 loops compared with the other three chains, creating binding site topologies comparable to those of mammalian TCR in basic paratope structure; additionally, nurse shark TCRδ CDR3 are more similar to IgH CDR3 in length and heterogeneity than to other TCR chains. Most interestingly, several cDNAs were isolated that contained IgM or IgW V segments rearranged to other gene segments of TCRδ and α. Finally, in situ hybridization experiments demonstrate a conservation of both α/β and γ/δ T cell localization in the thymus across 450 million years of vertebrate evolution, with γ/δ TCR expression especially high in the subcapsular region. Collectively, these data make the first cellular identification of TCR-expressing lymphocytes in a cartilaginous fish.

Macrophage-like effector of spontaneous cytotoxicity from the shark.

The effector of spontaneous cytotoxicity from shark peripheral blood has been shown to be a macrophage-like cell. Effector cells are isolated by centrifugation over Lymphocyte Separation Medium, adherence to glass, Percoll density gradient centrifugation and adherence to fibronectin sequentially. Effector cells are adherent to glass, sediment to densities of 1.048-1.052 g/ml and are adherent to fibronectin. The isolated effectors represent less than 1% of the peripheral blood leukocytes, and exhibit potent cytotoxic capability, both spontaneous and in the presence of phytohemagglutinin. In addition, the activity of these cells is resistant to 3000 rads gamma irradiation. Although nurse sharks have natural antibody to trinitrophenol, spontaneously cytotoxic cells are incapable of killing trinitrophenol modified targets indicating that natural antibody is not required for reactivity, and that natural antibody and spontaneous effectors do not have the same repertoire. However, cold target inhibition studies showed that these effector cells can recognize four of five human lymphomyeloid targets. It is concluded that the spontaneous, extracellular killing by the macrophage-like effector most closely resembles that of activated mammalian tumoricidal macrophages with the exception that they do not appear to require in vitro activation.

Damselfish with neurofibromatosis exhibit cytotoxicity toward tumor targets

Damselfish neurofibromatosis (DNF) is a malignant transmissible disease affecting Schwann cells, and is the only naturally occurring animal model of human neurofibromatosis type 1. The current study was designed to determine whether fish in the early stages of disease have measurable immune responses toward DNF tumor cells. Three DNF tumor cell lines were used as targets in standard 51Cr cytotoxic assays. In addition, Lutjanus griseus erythrocytes served as nonspecific targets, and concanavalon A (Con A) blasts from healthy animals served as normal target cells. Results of this study show that tumor-bearing damselfish have cells capable of destroying tumor targets but healthy animals display minimal, if any, reactivity toward the DNF tumor lines. The majority of antitumor activity resides in the spleen; the pronephros appears to contain the majority of nonspecific activity. Data also show that some of the effector cells are analogous to the nonspecific cytotoxic cells of catfish. No lysis of healthy damselfish targets was observed. Thus damselfish have cytotoxic cells capable of interacting with tumor targets, but in the majority of animals this response is not adequate to circumvent the process of tumorigenesis.

Sinus histiocytosis with massive lymphadenopathy. A case with unusual skin involvement and a therapeutic response to vinblastine‐loaded platelets

A patient with sinus histiocytosis with massive lymphadenopathy had unusual skin lesions and progressive internal involvement. Both aspects responded dramatically to the administration of vinblastine‐loaded platelets. Vinblastine‐loaded, idiopathic thrombocytopenic purpura antibody‐coated platelets are a rationale therapy for a disease characterized by the presence of actively phagocytosing histiocytes.

Fc receptor for shark IgM

Fc receptors for shark IgM have been demonstrated on shark leukocytes. Measurement of receptor binding required treatment of leukocytes with Cytochalasin D to inhibit phagocytosis. EA rosetting assays were carried out using human erythrocytes coated with shark anti-human antibody. Binding to shark leukocytes was demonstrated to be specific to shark IgM in that affinity purified shark IgM and purified Fc5 mu fragments could block rosette formation, but not shark transferrin, bovine serum albumin or fetal bovine serum. The binding was shown to be saturable and reversible, characteristic of receptor-ligand interaction. Further, it was shown that affinity purified, radioiodinated IgM could also bind Cytochalasin D-treated shark leukocytes in a manner analogous to rosetting. We conclude that Fc receptors appeared early in evolution, and that previous difficulties in demonstrating the Fc mu receptor resulted from non-specific binding associated with phagocytosis.

Mitogen induced cytotoxicity in the nurse shark

Abstract Nurse shark ( Ginglymostoma cirratum ) leukocytes isolated from peripheral blood were demonstrated to possess cytotoxic activity towards xenogeneic marine erythrocyte targets (grey snapper) when stimulated by phytohemagglutinin, concanavalin A, or lipopolysaccharide. These leukocytes when separated on the basis of adherence to glass exhibited a differential cytotoxic effect. Only the adherent population showed significant cytotoxicity toward the target cells in the presence of any of the mitogens. Wright-Giemsa staining showed some differences in the two populations, however, both adherent and nonadherent cell populations contain all types of leukocytes. Upon surface marker analysis, the adherent cells were found to be depleted of sheep erythrocyte rosette forming cells and surface immunoglobulin positive cells compared to the nonadherent and unseparated populations. The cytotoxic reaction in the shark resembles that of mammalian species and the effector leukocyte(s) may be the phylogenetic precursor of mammalian cytotoxic effector cells.

Damselfish with neurofibromatosis exhibit cytotoxicity towards retrovirus infected cells

Lymphocytes from tumor-bearing damselfish are cytotoxic towards target cell lines derived from damselfish neurofibromatosis. These cell lines contain at least one retrovirus which appears to be related to the etiology of the disease. The current studies were designed to characterize the effectors of this cytotoxic reaction. Data presented here show that cells separated using an antibody (5C6.10.4) directed towards non-specific cytotoxic cells of catfish sequesters all antitumor activity in the 5C6.10.4 negative population. Thus, damselfish 5C6.10.4 positive cells bind to tumor targets, but do not contribute to target cell death. In contrast, 5C6.10.4 positive cells are cytotoxic towards xenogeneic erythrocytes. Cytotoxicity of splenocytes from animals inoculated with virus purified from the 88-503 cell line suggested that prior exposure to the retrovirus enhanced reactivity, especially towards 88-503. In addition, cytotoxicity was significantly greater in tumor homogenate injected animals that resisted tumor development for more than 5 months as compared to those that developed tumors quickly. Lastly, cytotoxic responsiveness towards primary cultures of mock and virus infected autologous and allogeneic cells implies that the cytotoxic effector is directed towards retrovirus infected cells.

Allelic polymorphism of T-cell receptor constant domains is widespread in fishes.

T-cell receptor chains contain membrane-proximal constant domains of the immunoglobulin superfamily that are relatively invariant in mammalian species. In contrast, recent studies in the bicolor damselfish have demonstrated surprising allelic polymorphism in the TCR alpha (A) and TCR beta (B) “constant” (C) domain genes. This report extends these initial observations beyond Perciformes to two other orders of teleost fishes. Studies in both the Atlantic cod and zebrafish show high levels of polymorphism in the TCRA constant genes. Levels of 13% and 15% amino acid nonidentity were found within cod and zebrafish, respectively. Evolutionary analysis of codon usage suggests that positive selection maintains the high number of TCRAC alleles in these fish populations. Additionally, investigation of a TCRB constant gene from the Beau Gregory, a sister species of the bicolor damselfish, shows no evidence of transpecies maintenance of constant region alleles. These data argue that the T-cell receptor constant domain is being employed by many vertebrates in a manner inconsistent with our current understanding, and may indicate unheralded complexity in signal transduction through the TCR/CD3 complex.