Elizabeth A. Stein

The Role of Opsonins in Phagocytosis by Coelomocytes of the Earthworm, Lumbricus Terrestris

Abstract The effects of coelomic fluid factors on phagocytosis of yeast by coelomocytes of the earthworm, Lumbricus terrestris , were studied in vitro, examining both whole coelomocyte populations and specific coelomocyte types. Phagocytosis by basophils, the predominant coelomocyte type, and by granular cells (acidophils, granulocytes and chloragogen cells) was not enhanced by coelomic fluid factors. Neutrophils, however, exhibit enhanced phagocytosis in diluted coelomic fluid or after pre-opsonizing yeast with coelomic fluid. Undiluted coelomic fluid exerts a partial inhibiting effect on both phagocytosis and adherence by all coelomocyte types. Measurements of phagocytosis in whole coelomocyte populations show little evidence of opsonization by coelomic fluid, probably because of the relatively small numbers of neutrophils in the coelomocyte population.

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 earthworms humoral factors that may participate in immune responses.

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.

Cytochemical observations of coelomocytes from the earthworm,Lumbricus terrestris

SynopsisCoelomocytes of the earthworm,Lumbricus terrestris, were stained by cytochemical techniques to determine the biochemical composition of the seven different cell types and subtypes. The enzymes acid phosphatase and β-glucuronidase are present in all types of coelomocytes, but are especially abundant in basophils and neutrophils; the differences in enzyme amounts correlate well with the differences in phagocytic activity of the various cell types. No peroxidase is present. The cytoplasmic basophilia of basophils is due primarily to ribonucleic acid. Basophils also contain large deposits of glycogen, with neutrophils and chloragogen cells containing somewhat lesser amounts. The predominant granules of the two types of acidophils and of granulocytes are composed of a basic protein and a neutral mucopolysaccharide or glycoprotein. A second granule population, present in low numbers in acidophils and granulocytes, but in larger numbers in basophils and neutrophils, is small in size and lipid-positive and may, in part, represent lysosomes.Lipid is especially abundant in the vesicles and granules of the two types of chloragogen cells. Some granules of chloragogen cells also contain ferrous and ferric iron and a substance with pseudoperoxidase activity. The cytoplasm contains protein, glycogen, and a neutral mucopolysaccharide. In addition, acid mucopolysaccharides are variably present in the cytoplasm of chloragogen cells, the only coelomocytes to contain this class of substances.

Prevention of overt diabetes and insulitis by intrathymic injection of syngeneic islets in newborn nonobese diabetic (NOD) mice.

Based on previous studies showing that allogeneic islets transplanted into the thymus can induce donor-specific unresponsiveness, we investigated in the nonobese diabetic (NOD) mouse the effect of intrathymic islet isografts on preventing autoimmunity directed against pancreatic islet antigens. Islets prepared from newborn NOD pancreata were injected into one lobe of the thymus of 10− to 11-day-old female NOD mice (experimental group) with no immunosuppression. PBS alone was used for injection into age- and sex-matched litter mates (control group). Thirty of 32 (94%) experimental mice remained normoglycemic for over 30 weeks. Well-formed islets with no indication of insulitis were found in the thymus of these 30 mice, whereas no grafted islets were found in the 2 mice that became diabetic at 17 and 19 weeks, respectively (technical failures). In the control group, 10 of 32 (31%) mice became diabetic between 20 and 29 weeks. This diabetic incidence was, however, lower than that in our colony female mice. In the pancreas of experimental mice, 90.9% of islets were free of infiltrates, whereas only 13.1% of islets were intact in control mice. The spleens of 30-week-old experimental mice contained a slightly higher percentage of CD8+ T cells (P< 0.05) than those of control mice. Cyclophosphamide injections at 30 weeks induced diabetes in 4 of 9 experimental mice. The 2 lines of evidence, (1) marked reduction in insulitis of intrathymic islet-grafted mice and (2) induction of diabetes after treatment with cyclophosphamide, suggest that both thymic clonal deletion and peripheral tolerance may play a role in preventing diabetes.

Mitogenic Effect of Earthworm (Lumbricus terrestris)Coelomic Fluid on Mouse and Human Lymphocytes

We have cultured mouse and human lymphocytes with earthworm (Lumbricus terrestris) coelomic fluid and measured their mitogenic responses. Normal fluid was collected from untreated worms, while induced fluid was harvested from worms injected 24 h earlier with rabbit erythrocytes. At low protein concentrations in the coelomic fluid, human and mouse lymphocytes were significantly activated, as measured by incorporation of 3HTdr. The activation index for induced fluid was approximately 2.5 times that for uninduced fluid. Separation of mouse lymphocytes into B- and T-cell populations revealed that primarily T cells were activated. Normal and induced coelomic fluid contains 12 and 28 electrophoretic bands and agglutinin titers of 32 and 1024, respectively. Addition of agglutinin inhibitors or absorption of agglutinins from coelomic fluid did not alter levels of mitogenic activity, thus the relation between earthworm agglutinins and mitogens is problematic. Techniques designed to separate and purify the agglutinins are in progress to elucidate this point.

Swine as an allotransplantation model

Because of their anatomical and physiological similarities to humans, pigs are well suited for solving technical problems associated with clinical transplantation. The availability of genetically defined strains of miniature swine is unique with this species, and has permitted the investigation, in a large animal model, of immunological and genetic aspects of allograft rejection, clinical manifestations associated with bone marrow transplantation, and the feasibility of various approaches for induction of transplantation tolerance. Immunological assays and various reagents are also available in order to pursue these studies.

Bacterial agglutinins of the earthworm, lumbricus terrestris

Abstract 1. 1. Coelomic fluid of the earthworm, Lumbricus terrestris , contains agglutinins that bind with and agglutinate both Gram-positive and Gram-negative bacteria. 2. 2. Agglutinins were tested with five different bacterial strains, three of them isolated from Lumbricus coelomic fluid. 3. 3. When worms were injected with bacteria, four of the five stains induced higher levels of coelomic fluid agglutinins within 24 hr. 4. 4. Each of the bacterial strains reacted with different agglutinins or combinations of agglutinins, based on analyses of absorption, carbohydrate inhibition and heat inactivation data. 5. 5. KDO, 2-keto-3-deoxyoctonate, was the only sugar to consistently inhibit agglutination of all five bacterial strains by both naturally occurring and induced agglutinins.

Carbohydrate and glycoprotein inhibitors of naturally occurring and induced agglutinins in the earthworm, Lumbricus terrestris

Abstract 1. 1. Agglutinins in the coelomic fluid of Lumbricus terrestris are inhibited by a number of glycoproteins and polysaccharides. Naturally occurring agglutinins are inhibited most strongly by bovine submaxillary mucin and fetuin, and induced agglutinins by thyroglobulin, bovine submaxillary mucin, hyaluronic acid and fetuin. 2. 2. The only simple sugar to be specifically inhibitory was 2-keto-3-deoxyoctonate, a monosaccharide found in bacteria. 3. 3. Desialation of erythrocytes resulted in increased agglutination titers, possibly by allowing easier access of agglutinins to underlying carbohydrates. 4. 4. Positively charged substances reduced agglutination in proportion to their strength of charge, probably a non-specific effect. 5. 5. Inhibitor data suggest that the combining site of Lumbricus agglutinins may normally accommodate several sugars.

Separation and partial purification of agglutinins from coelomic fluid of the earthworm, Lumbricus terrestris

Abstract 1. 1. Agglutinins from coelomic fluid of the earthworm, Lumbricus terrestris were separated by gel chromatography. Fluid from erythrocyte-injected (induced) worms contained five agllutinin peaks with molecular wts ranging from 4000 to 400,000, whereas fluid from untreated (naturally occurring) worms contained only one agglutinin peak of mol. wt 30,000. 2. 2. Gel chromatography-separated agglutinin from naturally occurring fluid was unaffected by heating at either 60 or 100°C. Three of the agglutinins from induced fluid were unaffected by heating whereas two were reduced in titer by 36.5 to 88%. 3. 3. Affinity chromatography of induced coelomic fluid using bovine submaxillary mucin (BSM), conjugated affigel 15, eluted with potassium thiocyanate (KSCN), resulted in a purification factor of 105-fold, while bovine submaxillary mucin-Sepharose, eluted with 1 M NaCl, purified agglutinin(s) 93-fold.