Charles H. Bigger

Characterization of a C3-like cDNA in a coral : phylogenetic implications

C3, C4, and C5 are thiolester-containing proteins (TEPs) of vertebrate complement. The identification of the molecular origin of the TEP family, and more specifically the ancestor protein of complement components C3, C4, and C5, remains a fundamental question. The prevailing paradigm suggests that duplication and divergence of these proteins occurred after the deuterostome split in phylogeny. It is believed that the ancestor of thiolester-containing complement proteins was alpha-2-macroglobulin (A2M)-like, a noncomplement-related protein. Here we describe a C3-like cDNA from a gorgonian coral, Swiftia exserta. This study provides evidence for the origins of a complement-related C3-like gene in the Precambrian period, predating both protostomes and deuterostomes. Furthermore, one may speculate that complement-like opsonic reactions were evolving at the earliest stages of metazoan evolution. This calls for a reassessment of the present concepts concerning the origins and evolution of TEPs.

INTERSPECIFIC AND INTRASPECIFIC ACRORHAGIAL AGGRESSIVE BEHAVIOR AMONG SEA ANEMONES: A RECOGNITION OF SELF AND NOT-SELF

The acrorhagial responses of four sea anemones, Anthopleura krebsi, Bunodosoma cavernata, Anemonia sargassensis, and Anthopleura xanthogrammica, are described. All four acrorhagial responses can be considered forms of aggression. The acrorhagial response is only one of several responses of sea anemones to contact with other animals; others include several methods of avoidance and feeding. Prior experience can influence the acrorhagial response. In A. krebsi, the effect of a prior encounter on the excitation threshold can be seen for at least 2 hr.Interspecific behavioral interactions were examined in A. krebsi, B. cavernata, and A. sargassensis. With one exception, acrorhagial responses were only elicited by contact with some anthozoans. The exception is that some A. krebsi respond to the scyphistomac of the scyphozoan Cassiopea. xamachana. Some C. xamachana medusae from the same clone also elicited acrorhagial expansion and application behavior but never acrorhagial peeling.Intraspecific interactions wer...

Characteristics of transplantation immunity in the sponge, Callyspongia diffusa.

Very extensive polymorphism of histocompatibility (H) molecules in the sponge Callyspongia diffusa was revealed by the incompatibility found among 480 different allogeneic pairings without exception. This represents some 960 individual clones utilized as alloparabionts or sources of fitted allografts. Consistent cytotoxic reactivity provoked by allogeneic cell surface contact in Callyspongia is manifest in a continuum of strong to weak histoincompatibilities; however, strong reactions are most frequent at normal temperatures of 23 to 27 C. The directionality, vigor, and timing of alloparabiotic rejection in this species all depend mainly on the genetic constitutions of the interacting clones. Replicate interclonal combinations yield essentially identical rejection reactions, and differences in graft dosage had no effect on either the direction or intensity of allocytotoxicity. Alloreaction times are inversely proportional to the strength of the H barrier. Maximal allosensitization indicated by accelerated second-set reactivity was gradually achieved, as a function of the interclonal combination, after 2 to 8 days of primary contact. Heightened transplantation immunity or memory persists for at least 2 to 3 weeks after contact sensitization.

Allogeneic polymorphism and alloimmune memory in the coral, Montipora verrucosa.

Populations of the coral Montipora verrucosa exhibited very extensive allogeneic polymorphism revealed by parabiotic incompatibility among 890 different pairings of colonies (clones) without exception. More than 1,400 reciprocal histoincompatibilities were actually recorded because bilateral cytotoxicity was observed among most of the alloparabionts tested. This “uniqueness of the individual” in terms of cell surface histocompatibility (H) markers is manifest in the whole spectrum of possible incompatibilities from strong to moderate to weak. The directionality, severity, and rate of rejection were mainly determined by the genetic constitutions of the interclonal parabionts. Allograft reaction times were closely temperature dependent and inversely related to the strength of the H barrier. Heightened alloimmunity or early memory was demonstrable after only 2 days of presensitization and became maximal after 4 to 8 days. After prolonged primary immunization, potent alloimmune memory persisted for 4 weeks, but disappeared after 8 to 16 weeks. Immunological primitiveness in corals may therefore be reflectd in the lack of long-lived memory as found among vertebrates.

Aspects of Histocompatibility and Regeneration in the Solitary Reef Coral Fungia scutaria

Discoid coralla of the solitary free-living reef coral Fungia scutaria were cut with a rock saw and rejoined in various paired combinations and orientations of autogeneic sections (self to self), isogeneic sections (clone-mate to clone-mate), and allogeneic sections (two different genotypes). Results of these experiments provide the first evidence of histocompatibility in a solitary coral. Autogeneic or isogeneic sections of coralla with one section containing a mouth were joined along cut edges. In all cases, fusion of tissues occurred within weeks, followed by skeletal fusion within months. However, autogeneic or isogeneic sections rejoined along the uncut edges did not fuse. Isogeneic pairings between two sections with mouths produced neither tissue/skeletal fusion nor cytotoxicity at the interface. Individual cut sections were allowed to regenerate. Sections containing the parent mouth did not develop new mouths. However, cut sections lacking a mouth always regenerated multiple mouths along the cut edge, but not along the uncut edge. Sections without mouths cut along a second line parallel to the first cut always regenerated mouths along the cut edge located closest to what had been the mouth area of the original corallum. The new mouths eventually developed into individual polyps.

CYTOTOXIC TRANSPLANTATION IMMUNITY IN THE SPONGE TOXADOCIA VIOLACEA

The Hawaiian sponge, Toxadocia violacea, exhibited discriminating transplantation immunity in an extensive series of allogeneic and xenogeneic parabioses. Cytotoxic alloincompatibility occurred without exception, but with differing degrees of severity. The allorejection reactions developed more rapidly than has been observed in any other animal species. An intense tissue-bridging reaction in allogeneic contact zones preceded acute soft tissue destruction; the cytotoxic reactivity was usually restricted to the immediate zone of direct contact. The median reaction times were closely temperature-dependent and much accelerated at higher temperature. Specific, short-term alloimmune memory was revealed by second-set and third-party graftings. Extensive allogeneic polymorphism was evident from the invariable rejection observed among more than 140 different pairings. Xenograft reactions between Toxadocia violacea and Callyspongia diffusa were characterized by acute cytotoxicity, immune memory, and a qualitative change in secondary versus primary responses.

AFFERENT SENSITIZATION AND EFFERENT CYTOTOXICITY IN ALLOGENEIC TISSUE RESPONSES OF THE MARINE SPONGE CALLYSPONGIA DIFFUSA

Allogeneic branches of the marine sponge Callyspongia diffusa were paired either in parabiosis or as allografts. To examine the specificity of the allogeneic rejection process, naive second sets and third parties were substituted in the midst of primary allogeneic tissue responses. Other experiments were conducted with 0.2-, 0.8-, 3.0-, and 5.0-μm pore size membrane barriers separating various combinations of naive and presensitized allogeneic paired sponges. The results of these experiments demonstrate that both the afferent and efferent phases of allograft rejection in Callyspongia diffusa depend on direct allogeneic cell contact. Cytotoxic reactivity does not involve a readily diffusible cytotoxic molecule. Moreover, the specificity of allogeneic sponge graft rejections resides in the early recognition or afferent phase; once the effector response is generated or turned on, subsequent allogeneic cytotoxicity is nonspecific.

Alloimmunity in the Gorgonian Coral Swiftia exserta

This study of histocompatibility demonstrates that the gorgonian Swiftia exserta (Coelenterata, Anthozoan) fulfills the minimal functional criteria of cytotoxicity, specificity, and altered secondary response (memory) that characterize an adaptive immune response. All autografts (self grafts) fused, and all allografts (intraspecific grafts) underwent rejection, which is characterized by rapid and progressive blanching, necrosis, and loss of tissue in the immediate contact area. Initial reactions required 7-9 days to produce 1 mm of necrosis, but after a resting period, a second contact at a new tissue area yielded the same reaction in 3-4 days. After primary sensitization, intervals of up to eight weeks still produced a significantly accelerated secondary response. Significant differences between the reaction times of second set and third party allografts demonstrated recognition specificity in these responses. Thus, this is the first report of an adaptive alloimmune response in gorgonians.

Fetal pancreas transplantation in miniature swine. I. Developmental characteristics of fetal pig pancreases.

The genetically defined miniature pig developed by Sachs et al. was selected as a large animal model to test the feasibility of fetal pancreas transplants for reversal of insulin-dependent diabetes. In order to test our approach, the first key was to characterize the development of the pig pancreas tissues throughout fetal life. Pancreas samples were obtained from 102 farm pig fetuses ranging in age from 35–110 days and from 39 minipig fetuses removed by Caesarean section from 5 timed-pregnant sows between 33 and 73 days after conception. The development of the endocrine and exocrine pancreases were examined by immunobiochemical assays of insulin, chymotrypsinogen, and trypsinogen. Light and electron microscopic examination of pancreases from the critical fetal ages (35–55 days) were used to confirm the above results. Insulin was already present at day 33 and increased rapidly till birth (day 114: 2.2 U/pancreas). Chymotrypsinogen activity was first detected at day 43 and trypsinogen activity at days 49–50. Enzyme content increased rapidly till days 65–70 and then more gradually until birth. Morphological development of exocrine cell granules conformed to the above results. The results clearly demonstrated in the pancreas of a larger mammal, the pig, that the endocrine elements mature prior to the exocrine system. Thus, as we found in rats, pig fetal pancreas also has this advantage as a donor tissue for transplantation. An appropriate fetal age for pig donors is estimated to be between 45 and 50 days.

Historecognition and Immunocompetence in Selected Marine Invertebrates

All marine invertebrates must deal with pathogens, parasites and transformed cells. Sessile marine invertebrates must also meet the additional challenge of coming into contact with allogeneic and xenogeneic tissues. All invertebrate phyla examined demonstrate some degree of specific nonself reactions. Some sponges, scleractinian corals, gorgonians, echinoderms, and tunicates have demonstrated the existence of an alloimmune system by fulfilling the three functional criteria of: (1) cytotoxic reactions; (2) specificity; and (3) an inducible memory. Marine invertebrate immunologic memory is relatively short term, up to six months, but that could be significant, at least within the parameters of the animal’s life style. Tissue fusions require a positive recognition of self and the separate systems for allogeneic or xenogeneic tissue rejection require a positive recognition of nonself. Medawar’s concept of the “uniqueness of the individual” seems to be widely applicable but other systems/situations may occur. Hildemann’s proposal that cell mediated immunity arose early in metazoan evolution and Ig mediated responses appeared early in the vertebrates seems to be serving as a good working hypothesis. There are a number of other histoincompatibility systems in sessile marine invertebrates, e.g., sea anemone aggression, that share features with the alloimmune responses and could perhaps utilize related recognition molecules.