Specific tissue graft rejection in earthworms.

Earthworms are capable of destroying antigenic tissues. Autogeneis transplants healed in regularly and remained permanently viable. Xenografts, by contrast were cicatrized but eventually rejected. Intrafamilial transplants survived longer than interfamilial ones. Xenografts and autografts placed in the graft bed were joined to each other but xenografts were later destroyed although autografts were not. Two xenografts from Eisenia to Lumbricus, performed simul taneously, showed survival endpoints similar to a single xenograft. A 5-day interval between first- and second-set grafting led to an accelerated rejection of both transplants. First-set Allolobophora transplants to Lumbricus performed simulataneously with second-set Eisenia grafts were destroyed at a time different from either of the two Eisenia transplants. A single Allolobophora transplant to Lumbricus was rejected at survival tines equivalent to Allolobophora along with two Eisenia transplants.

Anti-inflammatory and anti-pyretic activities of earthworm extract-Lampito mauritii (Kinberg).

AIM OF THE STUDY: Experiments were conducted to understand the therapeutic properties such as anti-inflammatory and anti-pyretic activities of biologically active extract isolated from whole earthworm (Lampito mauritii, Kinberg). MATERIALS AND METHODS: Inflammation in the hind paw of Wistar albino rat, Rattus norvegicus, was induced by histamine, granuloma pouch was induced by turpentine and pyrexia induced by Brewer's yeast in rats were followed as earlier studies. Anti-inflammatory drug-indomethacin and anti-pyretic drug-paracetamol were used as standard drug for comparison. RESULTS: Administration of indomethacin (10mg/kg), paracetamol (150 mg/kg) and/or different doses of earthworm extract (EE) (50, 100 and 200mg/kg) reduced and restored to normal conditions in a dose-dependent manner of histamine and turpentine induced inflammation, and Brewer's yeast induced pyretic in rats. CONCLUSIONS: The most significant inhibition of paw oedema and granuloma and also the significant reduction in hyperpyrexia in rats when treated with standard drugs as well as different doses of EE, reflect the presence of anti-inflammatory and anti-pyretic properties of EE similar to glycoprotein complex (G-90).

Blurring borders: innate immunity with adaptive features.

Adaptive immunity has often been considered the penultimate of immune capacities. That system is now being deconstructed to encompass less stringent rules that govern its initiation, actual effector activity, and ambivalent results. Expanding the repertoire of innate immunity found in all invertebrates has greatly facilitated the relaxation of convictions concerning what actually constitutes innate and adaptive immunity. Two animal models, incidentally not on the line of chordate evolution (C. elegans and Drosophila), have contributed enormously to defining homology. The characteristics of specificity and memory and whether the antigen is pathogenic or nonpathogenic reveal considerable information on homology, thus deconstructing the more fundamentalist view. Senescence, cancer, and immunosuppression often associated with mammals that possess both innate and adaptive immunity also exist in invertebrates that only possess innate immunity. Strict definitions become blurred casting skepticism on the utility of creating rigid definitions of what innate and adaptive immunity are without considering overlaps.

Earthworm paste (Lampito mauritii, Kinberg) alters inflammatory, oxidative, haematological and serum biochemical indices of inflamed rat.

Experiments were conducted to understand the therapeutic properties such as anti-inflammatory, anti-oxidative, haematological and serum biochemical markers of earthworm paste (EP) derived from an indigenous species Lampito mauritii (Kinberg), in comparison with the standard anti-inflammatory drug- aspirin, on Wistar albino rat (Rattus norvegicus). Administration of earthworm paste of Lampito mauritii at the rate of 80 mg/kg into albino rats which were induced of inflammation, was found to reduce inflammation, restore the levels of antioxidants-reduced glutathione, glutathione peroxidase, superoxide dismutase, catalase and thiobarbituric acid reactive substances, normalise the values of erythrocyte, leukocyte, differential levels of neutrophils, lymphocytes, eosinophils, haemoglobin and serum biochemical contents e.g., protein, albumin, glucose, cholesterol, acid and alkaline phosphatase, electrolytes e.g., sodium, potassium and chloride. The anti-inflammatory activity together with antioxidant property of EP seems to be due to the high polyphenolic content of earthworm tissue.

Anti-ulceral and anti-oxidative properties of "earthworm paste" of Lampito mauritii (Kinberg) on Rattus Norvegicus.

Studies have been made to understand the anti-ulceral and anti-oxidant properties of the "earthworm paste" derived from Lampito mauritii (Kinberg), an indigenous species, in comparison with the standard anti-ulceral drug-ranitidine, on the Wistar strain albino rats Rattus norvegicus. Administration of 200 mg/kg aspirin was found to increase the volume of gastric juice secretion, total acidity, free acidity, ulcer index and reduce the pH. It also had decreased the anti-oxidant levels such as reduced glutathione, glutathione peroxidase, catalase, superoxide dismutase and increased the level of thiobarbituric acid reactive substances. Pretreatment with the standard drug-ranitidine (50 mg/kg) and different doses of "earthworm paste" (20, 40, 80, 160 and 320 mg/kg) in ulcer induced animal had enhanced the pH, decreased the volume of gastric juice, free acidity, total acidity and reduced the ulcer index. Further the activities of reduced glutathione, glutathione peroxidase, catalase, superoxide dismutase were increased whereas the thiobarbituric acid reactive substance had decreased. The results were more significant in rats administered with 160 mg/kg "earthworm paste" than the application of ranitidine and other doses of "earthworm paste". This indicates the presence of antiulcer and anti-oxidative effects in "earthworm paste". In conclusion, administration of 160 mg "earthworm paste"/kg was found to have better therapeutic properties.

Still waiting for the toll?

Multicellular organisms including invertebrates and vertebrates live in various habitats that may be aquatic or terrestrial where they are constantly exposed to deleterious pathogens. These include viruses, bacteria, fungi, and parasites. They have evolved various immunodefense mechanisms that may protect them from infection by these microorganisms. These include cellular and humoral responses and the level of differentiation of the response parallels the evolutionary development of the species. The first line of innate immunity in earthworms is the body wall that prevents the entrance of microbes into the coelomic cavity that contains fluid in which there are numerous leukocyte effectors of immune responses. When this first barrier is broken, a series of host responses is set into motion activating the leukocytes and the coelomic fluid. The responses are classified as innate, natural, non-specific, non-anticipatory, non-clonal (germ line) in contrast to the vertebrate capacity that is considered adaptive, induced, specific, anticipatory and clonal (somatic). Specific memory is associated with the vertebrate response and there is information that the innate response of invertebrates may under certain conditions possess specific memory. The invertebrate system when challenged affects phagocytosis, encapsulation, agglutination, opsonization, clotting and lysis. At least two major leukocytes, small and large mediate lytic reactions against several tumor cell targets. Destruction of tumor cells in vitro shows that phagocytosis and natural killer cell responses are distinct properties of these leukocytes. This has prompted newer searches for immune function and regulation in other systems. The innate immune system of the earthworm has been analyzed for more than 40 years with every aspect examined. However, there are no known entire sequences of the earthworm as exists in these other invertebrates. Because the earthworm lives in soil and has been utilized as a successful monitor for pollution, there are studies that reveal up and down regulation of responses in the immune system after exposure to a variety of environmental pollutants. Moreover, there are partial sequences that appear in earthworms after exposure to environmental pollutants such as cadmium and copper. There are now attempts to define the AHR receptor crucial for intracellular signaling after exposure to pollutants, but without linking the signals to changes in the immune system. There are several pathways for signal transduction, including JAK/STAT, TOLL, TRAF PIP3, known in invertebrates and vertebrates. For resistance to pathogens, conserved signal transduction components are required and these include a Toll/IL-1 receptor domain adaptor protein that functions upstream of a conserved p38 MAP kinase pathway. This pathway may be an ancestral innate immune signaling pathway found in a putative common ancestor of nematodes, arthropods and even vertebrates. It could also help us to link pollution, innate immunity and transduction in earthworms.

Anticipating innate immunity without a Toll.

Earthworm innate immunity depends upon small and large leukocytes (coelomocytes) that synthesize and secrete humoral antimicrobial molecules (e.g. lysenin, fetidin, eiseniapore, coelomic cytolytic factor [CCF]; Lumbricin I). Small coelomocytes (cytotoxic) are positive (CD11a, CD45RA, CD45RO, CDw49b, CD54, beta(2)-m and Thy-1 [CD90]; CD24; TNF-alpha) but negative using other mammalian markers. Large coelomocytes (phagocytic) are uniformly negative. Specific earthworm anti-EFCC 1, 2, 3, 4 mAbs are negative for Drosophila melanogaster hemocytes and mammalian cells but positive those of earthworms. Coelomocytes contain several lysosomal enzymes involved in phagocytosis and a pattern recognition molecule (CCF) that may trigger the prophenoloxidase cascade a crucial innate immune response. Earthworms and other invertebrates possess natural, non-specific, non-clonal, and non-anticipatory immune response governed by germ line genes. Toll and Toll-like receptor signaling is essential for phagocytosis and antimicrobial peptide synthesis and secretion in insects and vertebrates but has not yet been shown to be essential in earthworm innate responses.

Structure and differential target sensitivity of the stimulable cytotoxic complex from hemolymph of the Mediterranean mussel Mytilus galloprovincialis.

A cytotoxic protein complex of 320 kDA was isolated from dialyzed plasma of the edible mussel, Mytilus galloprovincialis. Constituted by the assembly of several different proteins, the complex exhibits selective killing against eukaryotic cells, including erythrocytes, mouse tumor cells and protozoan parasites. High variability, which was not correlated with protein concentration, suggested that the immune response of naive mussels was in various stages of activation. Stimulation assays by different treatments in vivo resulted in significant increases in the activity of the plasma suggesting that cytotoxic complexes are involved in immune defense. Lytic activity appears to involve binding of cytotoxic complexes onto target cell membranes and the formation of transmembrane pores. This research provides more evidence that the innate immune system of invertebrates involves large cytotoxic proteins with a broad range of recognitive specificities in addition to small antibacterial, antifungal peptides.

Earthworm coelomocytes in vitro: cellular features and "granuloma" formation during cytotoxic activity against the mammalian tumor cell target K562.

Earthworms possess specific, adaptive, cellular immunodefense as well as non-specific responses found in other complex metazoans. Here we characterized coelomocytes from the earthworm Eisenia foetida by electron microscopy and cytofluorimetric analyses, and investigated structural changes that occur when effector coelomocytes and target K562 erythromyeloid human tumor cells interact during cytotoxic activity. In in vitro cultures 1) the two earthworm cell types (i.e. small and large coelomocytes) retained their morphological features; 2) their DNA content was significantly less than that of human lymphocytes and the erythromyeloid human tumor cell line K562; 3) significant percentages of coelomocytes were found to be in S or G2/M phases of the cell cycle. When cultivated alone for up to 3 h, coelomocytes formed no aggregates. However, when mixed with K562, coelomocytes spontaneously killed tumor cells, and cytotoxic reactivity was accompanied by the formation of multiple aggregates similar to granulomas. These results are the first to describe this type of earthworm non-specific "inflammatory" response in vitro against tumor cells.

Clavanins, alpha-helical antimicrobial peptides from tunicate hemocytes.

Hemocytes from the invertebrate Styela clava, a solitary tunicate, contained a family of four alpha-helical antimicrobial peptides that were purified, sequenced and named clavanins A, B, C and D. Each clavanin contained 23 amino acid residues and was C-terminally amidated. The tunicate peptides resembled magainins in size, primary sequence and antibacterial activity. Synthetic clavanin A was prepared and displayed comparable antimicrobial activity to magainins and cecropins. The presence of alpha-helical antimicrobial peptides in the hemocytes of a urochordate suggests that such peptides are primeval effectors of innate immunity in the vertebrate lineage.

Second-set allograft responses in the earthworm Lumbricus terrestris. Kinetics and characteristics.

Second-set Lumbricus terrestris allografts from the same donor to the same recipient undergo accelerated rejection if transplanted less than 10 days after first-set allografts. In contrast, second-set allografts, transplanted at longer intervals (20-90 days) show no accelerated rejection. Accelerated rejection of allotransplants under these kinetic conditions agrees with earlier intrafamilial xenotransplant results obtained in earthworms affirming an essential experimental variable: the time of second-set transplantation. Accelerated rejection, weak specificity, and short-term "memory" are three characteristics of the earthworm's allogeneic cellular defense/immune system. These reactions probably result from intense responses (although of short duration) of coelomocytes activated by first transplants that are still present at the time of second-set grafting but absent or inactive at later periods.

Killing of intrafamilial leukocytes by earthworm effector cells.

When Lumbricus and Eisenia coelomocytes are cultured together in intrafamilial xenogeneic combinations, significant cytotoxicity occurs at 24 h but not at 5 nor 72 h, as shown by trypan blue assay. In a 4.5-h assay, measuring 51Cr release, using an effector/target ratio of 25:1, unpooled cells from a single Lumbricus killed Eisenia cells at levels of 6% and 14%. However, Eisenia coelomocyte survival was high and identical in either cell-free xenogeneic (Lumbricus) coelomic fluid or in artificial medium. In this 1-way assay, earthworm (Lumbricus) coelomocytes act as effector cells that kill non-self target cells, even those of other earthworms. Comparisons with previous results reveal greater reliability and consistently repeatable results when the 51Cr release assay is used to measure cytotoxicity regardless of the targets.

In vitro agglutinin production by earthworm leukocytes.

Leukocytes of the earthworm, Lumbricus terrestris, secrete agglutinins in vitro, as shown by measuring agglutinin titers of the culture medium and by observing secretory rosette formation by leukocytes with erythrocytes. Leukocytes form the highest percentages of secretory rosettes with rabbit erythrocytes (RBC) and with other RBC species in the order: rat, guinea pig, mouse, calf, sheep, horse, goat. Leukocytes displayed allotypic specificity by forming rosettes selectively with erythrocytes from different individual rabbits. Eight sugars inhibited rosette formation, along with the polysaccharide mannan and the glycoproteins thyroglobulin and bovine submaxillary mucin. Cyclohexamide did not affect rosette formation, suggesting that agglutinins may be preformed and stored in leukocytes prior to secretion. Leukocytes also formed E-type rosettes with erythrocytes, but apparently utilized different receptors from those of secretory rosettes since they were not inhibited by the same sugars.

Inhibition of frog SK effector-target cell binding.

Substances known to inhibit mammalian NK cell activity during the first stage of lysis (i.e., effector cell-target cell binding) will also inhibit frog SK cell activity. We analyzed target cell lysis after conjugate formation between one target cell and at least one effector cell. Using frog SK effector cells and frog allogeneic and mammalian tumor target cells, we demonstrated that EDTA, the Ca+2 and Mg+2 chelating agent, but not EGTA, the Ca+2 chelating agent, inhibited binding. Thus, Mg+2 is required for conjugate formation. The inhibitory effects of EDTA were at least partially reversible following removal of EDTA. Binding also required membrane fluidity since pretreatment of targets with glutaraldehyde prevented effector cell binding. Adding glutaraldehyde after conjugate formation increased lysis. Modification of target cell surface proteins by DMSO and trypsin also inhibited binding of effector and target cells. Substances inhibiting binding decreased lysis as measured by 51Cr-release from target cells. Inhibition of binding between SK effector and target cells adds further support to our view that natural or spontaneous killing of foreign cells may be one of the most primitive immuno-defense mechanisms.

T-lymphocyte and B-lymphocyte dichotomy in anuran amphibians: II. Further investigations on the E-rosetting lymphocyte by using monoclonal antibody azathioprine inhibition and mitogen-induced polyclonal expansion.

We have continued to search for characteristics of T-lymphocytes in anuran amphibians. Inhibition assays using the monoclonal antibodies OKT-11 and T-11, the T-cell immunosuppressive drug azathioprine, and mitogens confirmed the nature of E-rosetting peripheral blood lymphocytes (PBL) in Rana pipiens. Both monoclonal antibodies specifically inhibited E-rosetting in a dose-dependent fashion; absorption of the monoclonals by frog PBL specifically removed inhibition on human E-rosetting T-cells. Lysostripping frog E-receptors with monoclonals and a second FITC antibody revealed a fluorescent lymphocyte population which capped off the receptor as shown by subsequent inhibition of E-rosetting. Significant resynthesis of E-receptors occurred 24 hours after lysostripping, especially in nylon wool effluent fractions. Azathioprine allowed us to define tentatively two subpopulations of E-rosetting lymphocytes: one nylon wool adherent and sensitive to inhibition at low concentrations and another resistant but sensitive to higher (cytotoxic) concentrations of azathioprine. In addition, T-cell but not B-cell mitogens induced expansion of E-rosetting cell populations. Our results support a thymic origin for Rana pipiens' E-rosetting cells. With respect to evolution, human and frog E-receptors are probably homologous, sharing at least one and probably two different antigenic determinants.

T-lymphocyte and B-lymphocyte dichotomy in anuran amphibians: III. Assessment and identification of inducible killer T-lymphocytes (IKTL) and spontaneous killer T-lymphocytes (SKTL).

We have established the existence of alloreactive inducible killer (IK) T-lymphocytes in Rana pipiens by injecting immunogenic concentrations of allogeneic frog erythrocytes (RBC). Assessment of specific IK activity was determined microscopically, observing effector-target conjugate formation, and spectrophotometrically as released hemoglobin (Hb) from lysed targets (RBC). The presence of spontaneous killer (SK) T-lymphocyte activity was also determined using unimmunized frogs and similar assay conditions. Assays using rabbit anti-frog Thy-1.1 antiserum inhibition, but not E-rosetted T-lymphocyte depletion, confirmed the T-lymphocyte category of both effector cell populations in Rana pipiens. For IK activity, we determined the 1) best priming doses, 2) best effector cell source (peripheral blood), 3) best priming route (intraperitoneal), 4) kinetics of immunity development, and 5) kinetics of lysis. Kinetics of lysis and organ distribution for spontaneous killer cells were also determined. Our results may assist 1) in establishing the evolutionary origin of cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells, and 2) in predicting where the capacity of immuno-surveillance against modified-self appeared in phylogeny. The implications are important for understanding origins of mechanisms of resistance against neoplastic conditions.

T-lymphocyte and B-lymphocyte dichotomy in anuran amphibians: I. T-lymphocyte proportions, distribution and ontogeny, as measured by E-rosetting, nylon wool adherence, postmetamorphic thymectomy, and non-specific esterase staining.

We used sheep erythrocyte (SRBC) E-rosetting, nylon wool fractionation thymectomy and nonspecific esterase staining to assess T-lymphocyte characteristics in Rana pipiens during various developmental stages. T-lymphocytes appear in the spleens of premetamorphic tadpoles. We found significant levels of lymphocytes classifiable as T-cells in the peripheral blood of adults, but fewer numbers of T-cells in the thymus, jugular bodies, spleen, pronephros, mesonephros, liver and bone marrow. In addition, thymectomized Xenopus laevis showed a sharp decrease in T-cells as evidenced by E-rosetting, ANAE stain and nylon wool fractionation. The presence of T-lymphocytes in anuran amphibians and the existence of a receptor for SRBC on a population of these cells, suggest conservation of T-cells during evolution.

Inhibition of SK cell activity in frogs by certain drugs and sugars.

We present similarities between mammalian natural killer (NK) cells and (anuran amphibian) frog spontaneous killer (SK) cells. A cytotoxic assay utilizing allogeneic erythrocytes as target cells was used and lysis assessed by measuring release of hemoglobin. SK effector cells, just as mammalian NK cells, are not sensitive to cycloheximide nor most simple sugars (50 mM glucose, glucose-6-phosphate, galactose, fucose, mannose). However, SK activity is inhibited by chloroquine, colchicine and mannose-6-phosphate. When SK cells were co-incubated with mammalian tumor cells, they were able to lyse only the NK-sensitive target YAC-1, but not other mammalian tumor cell targets including K562, Molt-4, Raji, P815 and EL4. Lysis of YAC-1 cells was also inhibited by colchicine and chloroquine. These results allow speculation on the evolution of cell mediated cytotoxicity since natural cytotoxic cells are present in ectothermic vertebrates.

Agglutinins in the earthworm Lumbricus terrestris: naturally occurring and induced.

A naturally occurring hemagglutinin against rabbit and rat erythrocytes is contained in the coelomic fluid of the earthworm Lumbricus terrestris. The hemagglutinin reacts with some chicken and human erythrocytes, but not others, and does not react with the erythrocytes of seven other vertebrates. Hemagglutinins appear in increased amounts within 24 hr after injection of rat, rabbit, horse and sheep erythrocytes, and some chicken and human erythrocytes, and are the highest (approximately four- to sevenfold) with rabbit erythrocytes. The response is brief, and increased or more rapid responses do not occur after multiple injections. Cross reactivity and absorption data indicate a close or possibly identical relationship between agglutinins induced against different erythrocyte types. Effects of heating, enzyme and chemical treatment on induced anti-rabbit erythrocyte agglutinins indicate at least two and perhaps three different agglutinins. Two of the agglutinins are protein, one trypsin-sensitive and the other trypsin-resistant. Agglutinin activity is reduced in the absence of divalent cations. Sensitivity to heat varies with the type of agglutinin. The naturally occurring agglutinin is a protein, trypsin-resistant and unaffected by heating at 100 degrees C for 30 min. These hemagglutinins constitute one of the earthworm's humoral factors that may participate in immune responses.

Characterization of a 14 kDa plant-related serine protease inhibitor and regulation of cytotoxic activity in earthworm coelomic fluid.

We have purified and characterized the serine protease inhibitor activity contained in the coelomic fluid of the earthworms, Eisenia. Serine protease inhibitor activity was stable between pH3 and 9.5, not flocculable by pH 3.0 and resistant to 100 degrees C for 15 min. or to 4 degrees C for 24 h. Ten microL of coelomic fluid was sufficient to inhibit in vitro the protease activity of 0.12 microgram of trypsin. Injection of living bacteria into earthworms resulted in increased serine protease activity 1-2 days post-injection, and increased serine protease inhibitor activity on day 4, suggesting that serine protease inhibitor is responsible for serine protease neutralization. Purified to homogeneity by affinity chromatography on trypsin, the serine protease inhibitor of Eisenia is a monomer of 14 kDa. Its partial NH2 amino acid sequence revealed a basic hydrophobic fragment which shared 68-75% homologies and 47-60% identities with several plant serine protease inhibitors. Eisenia cytotoxic activity due to the two fetidins of 40 and 45 kDa was stimulable in vitro by several serine proteases. Incubation with soybean trypsin inhibitor variant a (STIa) resulted in less cytotoxicity. The inhibitory effect occurred only when STIa was added before cell disruption. Interpretative cytotoxic scheme involving the release of intracellular cytotoxic proteins, intracellular trypsin-like activator and extracellular serine protease inhibitor suggests regulatory mechanisms for cellular/humoral immune system of earthworms.