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).

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.

Curcumin in cell death processes: a challenge for CAM of age-related pathologies.

Curcumin, the yellow pigment from the rhizoma of Curcuma longa, is a widely studied phytochemical which has a variety of biological activities: anti-inflammatory and anti-oxidative. In this review we discuss the biological mechanisms and possible clinical effects of curcumin treatment on cancer therapy, and neurodegenerative diseases such as Alzheimer's Disease, with particular attention to the cell death processes induced by curcumin. Since oxidative stress and inflammation are major determinants of the aging process, we also argue that curcumin can have a more general effect that slows down the rate of aging. Finally, the effects of curcumin can be described as xenohormetic, since it activates a sort of stress response in mammalian cells.

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.

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.

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.

Immune response to dietary proteins, gliadin and cerebellar peptides in children with autism.

The mechanisms behind autoimmune reaction to nervous system antigens in autism are not understood. We assessed the reactivity of sera from 50 autism patients and 50 healthy controls to specific peptides from gliadin and the cerebellum. A significant percentage of autism patients showed elevations in antibodies against gliadin and cerebellar peptides simultaneously. For examining cross-reaction between dietary proteins and cerebellar antigens, antibodies were prepared in rabbits, and binding of rabbit anti-gliadin, anti-cerebellar peptides, anti-MBP, anti-milk, anti-egg, anti-soy and anti-corn to either gliadin- or cerebellar-antigen-coated wells was measured. In comparison to anti-gliadin peptide binding to gliadin peptide at 100%, the reaction of anti-cerebellar peptide to gliadin peptide was 22%, whereas the binding of anti-myelin basic protein (MBP), anti-milk, anti-egg and anti-soy to gliadin was less than 10%. Further examination of rabbit anti-gliadin (EQVPLVQQ) and anti-cerebellar (EDVPLLED) 8 amino acid (AA) peptides with human serum albumin (HSA) and an unrelated peptide showed no binding, but the reaction of these antibodies with both the cerebellar and gliadin peptides was greater than 60%. This cross-reaction was further confirmed by DOT-immunoblot and inhibition studies. We conclude that a subgroup of patients with autism produce antibodies against Purkinje cells and gliadin peptides, which may be responsible for some of the neurological symptoms in autism.

Earthworm leukocytes kill HeLa, HEp-2, PC-12 and PA317 cells in vitro.

Earthworm coelomic fluid contains biologically active molecules and leukocytes that participate in phagocytosis, encapsulation. Presumably they synthesize and secrete several effector modulators of innate immune responses such as antibacterial molecules, cytotoxic proteins and cytokines. Several lytic molecules have been detected in coelomic fluid previously but it is not yet clear which are actually released from the coelomocytes. Our aim was to analyze the cytotoxic effects of coelomocytes on mammalian target cells and to provide evidence that the lytic factors originate from coelomocytes. Cell-free coelomic fluid, supernatants of short-term cultured coelomocytes, and lysates from coelomocytes--derived by mechanical and detergent extraction--were used in cytotoxicity assays performed on different mammalian standard tumor cell lines and mouse fibroblasts. We used native and denaturized (using proteinase K, and trypsin digestions, or heat-inactivation) coelomocyte lysates (CCL). The viability controls of targeted cells were made by measuring photometrically and analyzing by inverted microscopy. According to our results the coelomic fluid, the supernatant of cultured coelomocytes, and the CCL significantly decreased ratios of living cells compared to controls in a dose-dependent manner. Our experiments performed with CCLs suggest that coelomocytes are responsible for the productions of cytotoxic components presumably proteins.

Alternative sources of fibrinolytic, anticoagulative, antimicrobial and anticancer molecules.

The medicinal properties of earthworms in various remedies date back to 1340 A.D. and have been extended to other countries and cultures. Assays of tissue homogenates of earthworm (Eisenia foetida) have revealed a glycolipoprotein mixture referred to as G-90 that is composed of macromolecules with medical and pharmaceutical applications. There are several functions attributed to G-90: possession of several growth factors that: stimulate proliferation in cell cultures, contain an insulin like growth factor (IGF like), an immunoglobulin like growth factor (IgFG-like), possess two serine peptidases with a tyrosine code and epidermal growth factor (EGF). In contrast, G-90 exerts strong fibrinolytic and anticoagulative activity capable of lysing fibrin clots. Actions of these two properties are dependent upon concentration. Anti-coagulative activity also depends upon the kind of anticoagulants (G-90, PI, PII). G-90 can also act as antioxidant, exert antimicrobial activities in vitro and in vivo. The bacteriostatic effect is significantly greater for non-pathogenic species. Finally G-90 also participates in tissue regeneration and wound healing. Taken together, components of earthworms could be tested in certain clinical trials.

Earthworm leukocyte populations specifically harbor lysosomal enzymes that may respond to bacterial challenge.

Earthworm leukocytes (coelomocytes) are responsible for innate cellular immune functions such as phagocytosis and encapsulation against parasites and pathogens. Microbial killing results from the combined action of the phagocytic process with humoral immune factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), and various cytotoxic and antimicrobial molecules. There is also evidence of weak adaptive immune responses against foreign transplants. This study focused on aspects of the innate immune response. First, anti-human acid phosphatase (anti-AcP) polyclonal antibody characterized different acid hydrolase patterns in coelomocytes. Second, flow cytometry identified a strongly immunoreactive coelomocyte population. Third, ultrastructural and cytochemical analyses revealed acid phosphatase in discrete granules (lysosomes) of effector hyaline and granular coelomocytes but not in mature chloragocytes. Coelomocytes were exposed to bacteria to assess how phagocytosis influences: (a) the production of acid phosphatase using Western blot, and (b) release of acid phosphatase using ELISA from cell-free coelomic fluid. Fourth, after phagocytosis, acid phosphatase levels differed between controls and experimentals. Fifth, we found a 39-kDa molecule that reacted intensely with anti-AcP. Our results suggest that effector earthworm coelomocytes may not eliminate pathogens only by phagocytosis but also by extracellular lysis.

Infections, toxic chemicals and dietary peptides binding to lymphocyte receptors and tissue enzymes are major instigators of autoimmunity in autism.

Similar to many complex autoimmune diseases, genetic and environmental factors including diet, infection and xenobiotics play a critical role in the development of autism. In this study, we postulated that infectious agent antigens such as streptokinase, dietary peptides (gliadin and casein) and ethyl mercury (xenobiotic) bind to different lymphocyte receptors and tissue enzyme (DPP IV or CD26). We assessed this hypothesis first by measuring IgG, IgM and IgA antibodies against CD26, CD69, streptokinase (SK), gliadin and casein peptides and against ethyl mercury bound to human serum albumin in patients with autism. A significant percentage of children with autism developed anti-SK, anti-gliadin and casein peptides and anti-ethyl mercury antibodies, concomitant with the appearance of anti-CD26 and anti-CD69 autoantibodies. These antibodies are synthesized as a result of SK, gliadin, casein and ethyl mercury binding to CD26 and CD69, indicating that they are specific. Immune absorption demonstrated that only specific antigens, like CD26, were capable of significantly reducing serum anti-CD26 levels. However, for direct demonstration of SK, gliadin, casein and ethyl mercury to CD26 or CD69, microtiter wells were coated with CD26 or CD69 alone or in combination with SK, gliadin, casein or ethyl mercury and then reacted with enzyme labeled rabbit anti-CD26 or anti-CD69. Adding these molecules to CD26 or CD69 resulted in 28-86% inhibition of CD26 or CD69 binding to anti-CD26 or anti-CD69 antibodies. The highest % binding of these antigens or peptides to CD26 or CD69 was attributed to SK and the lowest to casein peptides. We, therefore, propose that bacterial antigens (SK), dietary peptides (gliadin, casein) and Thimerosal (ethyl mercury) in individuals with pre-disposing HLA molecules, bind to CD26 or CD69 and induce antibodies against these molecules. In conclusion, this study is apparently the first to demonstrate that dietary peptides, bacterial toxins and xenobiotics bind to lymphocyte receptors and/or tissue enzymes, resulting in autoimmune reaction in children with autism.

Dissociation of innate and adaptive immunity by UVB irradiation.

Increasing ultraviolet-B irradiation (UVB) resulting from diminution of stratospheric ozone is becoming a serious international problem. UVB irradiation exerts not only carcinogenic effects on animals but also causes them to become vulnerable to infections by modulating their immune responses. UVB irradiation suppresses innate immune functions of cells such as macrophages, neutrophils, Langerhans cells, dendritic cells, and the serum component, complement. UVB irradiation also causes changes in cytokine profiles, represented by the induction of a paradigm switch involving Th1/Th2 phenotypes. According to earlier studies, Th1 responses are suppressed, whereas Th2 activities are augmented by UVB irradiation. These immune modulations are caused by several pathways via cytokines and neuropeptides, and eventually may lead to increasing incidences of infection, allergy, and cancer. We have reviewed reports concerning UVB-irradiation induced immune modulation from the viewpoint of risks for human diseases and, in addition, for ecosystems and immunity of lower animals.

UVB irradiation suppresses cytokine production and innate cellular immune functions in mice.

We examined whether ultraviolet-B (UVB) irradiation (6 kJ/m2) alters cytokine production and other innate immune reactions by murine peritoneal macrophages and peripheral neutrophils. Along with these experiments, serum IgG levels were also assessed. In addition, using scanning electron microscopy (SEM) we observed macrophages that had been exposed to UVB in vitro. Results showed that UVB irradiation: (1) decreased IL-12 production while increasing IL-1alpha secretion from macrophages, but had no effect on IL-1alpha from neutrophils; (2) suppressed phagocytosis of macrophages but not of neutrophils; (3) diminished active oxygen production of macrophages but not of neutrophils; (4) had no effect on serum IgG levels; and (5) caused significant cell destruction of macrophages in vitro. These results suggested: (1) that UVB irradiation could induce characteristic suppression of innate immunity; (2) that innate cellular immunity was more susceptible to the effects of UVB irradiation than humoral immunity.

Evidence for perforin-like activity associated with earthworm leukocytes.

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coelomocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocytochemistry using light and transmission electron microscopy. A rat-anti-mouse-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualized by light microscopy. TEM analysis using immunogold showed a homogenous labeling in the cytoplasm of small coelomocytes. The highest number of immunogold particles was estimated in coelomocytes with many small cytoplasmic granules. Coelomocytes with large lysosomal granules were also labeled but less intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsible for other lytic activities. MALDI-MS revealed calreticulin usually associated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). Together, results strongly suggest the presence of putative perforin in earthworms. This in turn supports the hypothesis that perforin is a conserved component important in immune defense during evolution.

Preliminary evidence of modulating Th1 cytokine after allergen challenge.

We wanted to determine whether a paradigm switch in Th1/Th2 phenotypes in splenic lymphocytes could be induced in BALB/c mice after ultraviolet-B (UVB) irradiation and exposure to pollen. Results showed that UVB irradiation increased IL-1 but decreased IL-12 in vivo, and caused significant cell destruction of macrophages in vitro. We then gave mice cedar pollen intranasally, UVB irradiation (6kJ/cm2), and then splenic lymphocyte functions were examined. Results revealed that: 1) the level of IFN-gamma of splenic lymphocytes from UVB-irradiated mice was significantly decreased, especially in pollen-exposed mice; 2) UVB irradiation did not augment IL-4 levels; 3) IgE levels from UVB-irradiated mice did not increase. UVB irradiation (6kJ/cm2) did not induce Th2 response but suppressed Th1 cytokine, suggesting that Th1 could be more susceptible to UVB irradiation than Th2.

Different types of response to foreign antigens by leech leukocytes.

We used morphological and immunocytochemical approaches to characterize and to show the behavior of cells involved in leech inflammatory responses. Leeches were injected with bacterial lipopolysaccharide, fluoresceinated yeasts, sulfate spheres and ciliates (Protozoa). Shortly after injection, migrating cells appeared in the area of injection. The response of the cells occurred in relation to the injected micro or macro antigens. Each injection first provoked a migration of cells towards the non-self material. Afterwards, different responses (degranulation, phagocytosis, encapsulation, melanization) occurred. The migrating cells involved in these series of processes have a similar behavior and are characterized by CD markers of macrophages, NK cells and granulocytes, which are typical of many invertebrates and vertebrates.

Ontogeny recapitulates phylogeny: comparative immunology in Germany.

The innate immune system is in the spot light of modern immunology. Whenever protists, invertebrates and vertebrates are threatened by pathogens, they rapidly activate highly effective antimicrobial defense reactions. Because this young field develops very dynamically, it is important to ask what we really know about the mechanisms governing the innate immune defense system. This was the topic of a recent meeting entitled 'The Evolution of the Immune System', held at the Friedrich Schiller University in Jena, Germany. Leading scientists in the field of innate immunity presented their latest data in a historical and friendly setting.