Spermatogenesis in a nematode Nippostrongylus brasiliensis

The structure and development of the spermatozoon of Nippostrongylus brasiliensis was studied with the electron microscope using thinsectioned material and tissue prepared by the freeze-fracture technique.The developing germ cells are connected via a complex anucleate rachis which begins as fine threads of cytoplasm joining the spermatogonia. It rapidly enlarges to a broad, central core which not only anchors and joins the spermatocytes, but also appears to be an important site for protein synthesis. Formation of membranous organelies (MOs) from RER-associated Golgi bodies dominates the activities of the growing spermatocytes. As the latter approach meiosis, the rachis declines in importance and finally becomes the site of breakdown of the residual cytoplasm. The spermatid chromatin condenses into a long cylinder during spermatogensis. A pair of centrioles in an indentation at the anterior end are believed to organize long microtubules which are responsible for moving the nucleus through then out of the sperm cytoplasm to form a tail-like structure. Thus the cytoplasmic region of mature sperm contains only mitochondria and MOs; a small part of the anterior is amoeboid.     

Jejunal malabsorption in the rat infected by the nematode Nippostrongylus brasiliensis

Symons L. E. A., Gibbins J. R. and Jones W. O., 1970. Jejunal malabsorption in the rat infected by the nematode Nippostrongylus brasiliensis. International Journal for Parasitology, 1: 179–187. The rate of jejunal absorption of a range of substances absorbed actively or by diffusion was depressed in the rat infected by the nematode Nippostrongylus brasiliensis. The degree of malabsorption of actively absorbed substances was directly related to the severity of the infection.     

Characterization of mouse macrophage differentiation antigens by monoclonal antibodies

Monoclonal rat antibodies to mouse macrophage antigens were prepared. For immunization phagocytic cells in the spleens of mice recovering from sublethal irradiation were used. Specificities of the monoclonal antibodies obtained were determined on cells of normal mouse cell populations as well as on cells of a panel of mouse cell lines. In an attempt to monitor expression of differentiation-related antigens two models of in vitro-induced macrophage differentiation were used: differentiation of cells of the myeloblast line Ml; CSF-1-induced differentiation of bone marrow cells. The results obtained clearly show that during maturation from undifferentiated to highly differentiated cells of the macrophage lineage expression of antigens recognized by the MIV 38, MIV 55, MV 87, and MV 114 monoclonal antibodies is enhanced. At the same time, expression of antigens recognized by the MIV 52, MIV 113, and MIV 116 monoclonal antibodies diminishes at a similar rate. The suitability of these monoclonal antibodies for the characterization of differentiation states of mouse macrophages is discussed.     

Immunocytochemical localization of secretory acetylcholinesterase of the parasitic nematode Nippostrongylus brasiliensis

Various parasitic nematodes secrete acetylcholinesterase (AChE). In this study, the localization of AChE in the nematode Nippostrongylus brasiliensis and the secretory forms of AChE in culture fluid were examined. A thiocholine method revealed that AChE activity was localized in the subventral glands, which have a secretory and excretory function via a duct connected to the excretory pore. By electron microscopy, AChE activity was found mainly in the matrix of secretory granules, and sometimes in the Golgi apparatus in the subventral gland cells. These results show that nematode AChE is produced and stored in the subventral glands. Monoclonal antibodies against AChE of human erythrocytes or electric rays also bound to the nematode subventral gland, suggesting immuno-cross-reactivity of AChE among these species. When AChE activity in the nematode excretory-secretory product was examined by SDS polyacrylamide gel electrophoresis combined with the thiocholine method, intense activity was demonstrated as a single band at 74kDa. Immunoblot analysis showed specific recognition of this molecule by IgE and IgG1 antibodies, but not by IgG2a antibody, in nematode-infected rat sera. These results indicate that the nematode AChE molecule produced in and secreted from the subventral glands is antigenic for the production of IgE/IgG1 in host animals.     

Pyridoxal 5'-phosphate dependent enzymes in the nematode Nippostrongylus brasiliensis.

Eight classes of pyridoxal 5'-phosphate dependent enzymes have been investigated in Nippostrongylus brasiliensis in parallel with rat tissues. The range of decarboxylases detected in N. brasiliensis was limited in comparison with rat tissues. N. brasiliensis possessed a highly active L-serine hydroxymethyltransferase, but in contrast with rat liver, 5-aminolevulinic acid synthetase was absent. Similar levels of L-serine and L-threonine dehydratase activities were detected in N. brasiliensis and rat liver, and both organisms lacked L-alanine racemase, L-tryptophan synthetase and L-methionine gamma-lyase. The demonstration of cystathionine beta-synthase and gamma-cystathionase in N. brasiliensis suggests the presence of a functional trans-sulphuration sequence. The substrate specificities of the nematode cystathionine beta-synthase and gamma-cystathionase varied significantly from those of the corresponding mammalian enzymes. Particularly striking was the ability of N. brasiliensis cystathionine beta-synthase to catalyse the non-mammalian 'activated L-serine sulphydrase' reaction (L-cysteine + R-SH----cysteine thioether + H2S). N. brasiliensis and rat liver exhibited comparable abilities to transaminate amino acids via the 2-oxoglutarate: glutamate system.     

IgE synthesis in vitro during infection of mice with the nematode Nippostrongylus brasiliensis: effects of mitogens and antigens

In vitro IgE synthesis by lymphoid cells was studied during the course of infection of mice with Nippostrongylus brasiliensis. The studies involved inbred strains of mice which had been shown to be high IgE responders (A.CA, B10.M), or non-responders (Balb/c, B10.D2) to parasite antigen. In addition, F1 hybrids of low and high responders and irradiated non-responders were studied. Infection with N. brasiliensis led to an increase in IgE synthesis in vitro which was most pronounced during reinfection of mice. Addition of mitogens e.g. pokeweed mitogen (PWM), lipopolysaccharide (LPS), concanavalin A (ConA) to the cultures induced enhancement, suppression or had no effect on IgE synthesis. Addition of N. brasiliensis homogenate or worm culture supernatant led to a fluctuating pattern of IgE synthesis. No correlation was found between lymphocyte proliferative response to mitogen and worm antigens and IgE synthesis. Our data suggest, that PWM is more likely to enhance IgE synthesis in vitro than LPS or ConA. An enhancement is more easily observed with the cells of non-infected animals or during the early phase of infection or reinfection. The mitogen-induced increase in IgE synthesis did not exceed the values obtained during infection or reinfection.     

Characterization of human sperm surface antigens with monoclonal antibodies

Monoclonal antibodies (McAb) against human ejaculated sperm were developed from mice immunized with sperm membrane preparations. A solid-phase radioimmunoassay, with dried sperm as antigen, was employed in McAb screening. The tissue and species specificity of monoclonal antibodies HS 2, 4 and 6 were evaluated after absorption of antibody preparations with heterologous sperm, human serum or seminal plasma or cells from other human organs. The sensitivity of HS 2, 4 and 6 antigens to trypsin exposure was determined: HS 4 antigen was highly sensitive while HS 2 and 6 were not. The regional distribution of McAb 4 on intact sperm cells was determined by immunofluorescence staining. HS 4 may be a sperm-coating antigen based on its presence on sperm and in seminal plasma. This possibility led to an investigation of its role in sperm capacitation. HS 4 antibody binding was reduced when capacitated sperm were compared with noncapacitated cells. HS 4 antibody, when present during capacitation and insemination, was without effect on sperm motility or fusion with zona-free hamster eggs. Trypsin removal of as much as 60% of HS 4 antigen from the cell population also did not impact on sperm function. To identify the molecular correlate of HS 4 antigen, membrane components were extracted from washed sperm with Nonidet P-40, concentrated by acetone precipitation and analyzed electrophoretically in SDS-urea on 10% polyacrylamide slab gels. Immunoassays on protein blots with peroxidase-coupled second antibody identified a single reactive species in the molecular weight range of 130,000. Multiple reactive components were detected in blot transfers of seminal plasma.     

Characterization of baboon testicular antigens using monoclonal anti-sperm antibodies

Sperm antigens that appear during spermatogenesis in the baboon were identified by using three monoclonal antibodies generated in culture from mice immunized with baboon caudal epididymal spermatozoa. Antibodies BSA1 and BSA2 recognize trypsin-sensitive 84,000 and 45,000 dalton determinants that are restricted to the tail and anterior acrosomal regions of the sperm, respectively, as determined by Western blot and immunofluorescence techniques. The tail antigen absent in 2- and 3-yr-old baboon testes first appears in spermatid cells at about 4 yr of age. In contrast, the acrosomal antigen recognized by BSA2 is present in 3-yr-old primitive testicular germ cells. In the mature testis, the 45,000 molecular weight determinant is predominantly localized in the nucleus of late pachytene spermatocytes and round spermatid cells as observed via the avidinbiotin immunoperoxidase method. Antibody BSA3 reacted only with sailidase-treated sections of adult testis. This trypsin-resistant determinant, not expressed on testicular sperm, is recognized by antibody BSA3 only on epididymal sperm, thus indicating a post-testicular sperm modification.     

Hydrogen peroxide production in uncoupled mitochondria of the parasitic nematode worm Nippostrongylus brasiliensis.

1. Mitochondria from the parasitic nematode worm Nippostrongylus brasiliensis produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 2. Antimycin A inhibits respiration and H2O2 production by 70 and 65% respectively; the residual activities can be attributed to alternative electron-transport pathway(s). 3. o-Hydroxydiphenyl and 1,3,5-trihydroxybenzene, inhibitors of alternative electron transport, inhibit respiration by 37% and H2O2 production by 26%. 4. Another inhibitor of alternative electron transport, salicylhydroxamic acid, shows a complex mode of action; low concentrations (less than 0.5 mM) stimulate respiration and H2O2 production, whereas 2 mM-salicylhydroxamic acid inhibited respiration by 35% and stopped H2O2 production completely. 5. O2 thresholds were observed for the inhibition of respiration at O2 concentrations greater than 57.7 microM and inhibition of H2O2 production (greater than 20.5 microM-O2); apparent Km values for oxygen were 5.5 microM and 3.0 microM respectively. 6. In the presence of antimycin A the O2-inhibition thresholds and apparent Km values for O2 of respiration and H2O2 production matched closely, suggesting that the alternative oxidase is a likely site of H2O2 production. 7. These results are discussed in relation to O2 toxicity to N. brasiliensis.     

Characterization of Nippostrongylus brasiliensis infection in different strains of mice

Five mouse strains, CBA/J, BALB/c, C3H/HeJ, A/J, and C57Bl/6J-bg-bg, all showed similar expulsion kinetics for Nippostrongylus brasiliensis (infective dose = 500 L3). Typically, parasite recovery was maximal on day 2 in the lungs and by day 4 in the small intestine. Few worms (less than 5% infective dose) were recovered on day 14 in all strains. These same mouse strains exhibited immune depression on day 5 of infection with mesenteric lymph node cells (MLN) showing reduced (10-30% normal) IgM, IgG, and IgA responses against heterologous antigen. The intestinal mast cell numbers and tissue histamine levels were examined in CBA/J mice. Mast cell numbers increased (normal = less than 1/villous crypt unit; VCU) from day 5 and peaked on day 12 (greater than 15/VCU). Intestinal histamine levels did not completely correlate with mast cell numbers with maximum concentrations (240 +/- 73 ng/g, 2-fold over normal) reached by day 8. Histamine concentrations in the intestine returned to normal levels by day 20.     

Biogenic amines and gaba in the larval and adult forms of the nematode Nippostrongylus brasiliensis

1. Simultaneous detection (HPLC and electrochemical detection) of biological extracts of larval and adult stages of Nippostrongylus brasiliensis was performed in order to assay biogenic amines.2. Gamma-amino-butyric acid was assayed in the same samples.3. Tryptophan, 5-hydroxyindoleacetic acid were at the same level in adults and larvae.4. 5-Hydroxytryptophan, serotonin, dihydroxyphenylalanine and dopamine were significantly higher in larvae in which gamma-amino-butyric acid was not detected.     

Characterization of Chlamydia trachomatis antigens with monoclonal and polyclonal antibodies

We used monoclonal antibodies (MAbs) to examine the antigenic specificity and biologic function of several Chlamydia trachomatis antigens. Thirteen distinct MAbs to eight C. trachomatis antigens were produced. Six MAbs reacted with unique epitopes on the major outer membrane protein (MOMP) and two of these had neutralizing activity. MAbs were produced to each of the chlamydial antigens with molecular masses of 10, 29, 32, 57, 60, 70, and 75 kilodaltons (kDa). These MAbs showed species and genus specificity in an immunoblot assay. None of the MAbs had neutralizing activity. The epitopes recognized on MOMP, 29-, and 10-kDa (presumably lipopolysaccharide) antigens were surface exposed. MAbs to the 75-kDa, 57-kDa, and MOMP antigens were used for immunoaffinity purification of these antigens to produce monospecific antisera in mice. With polyclonal sera, we found that the 75-kDa antigen was also immunoaccessible and that antibody to MOMP and 75-kDa antigens neutralized C. trachomatis infectivity. We conclude that, in addition to MOMP and lipopolysaccharide, antigens with molecular masses of 75 and 29 kDa are surface exposed. Antibodies to MOMP and 75-kDa antigens can neutralize the organism in vitro.     

Ultrastructure of the hypodermis during cuticle formation in the third molt of the nematode Nippostrongylus brasiliensis

Summary The fine structure of the hypodermis of Nippostrongylus brasiliensis, a parasitic nematode of the rat, was studied in free-living third-stage filariform (infective) larvae, and in parasitic forms undergoing the third molt. In filariform larvae the hypodermis displayed a comparatively poorly developed RER, and few Golgi complexes, mitochondria, and vesicles. Nuclei contained large amounts of heterochromatin and no nucleoli. After worms reached the lungs and the third molt began, the RER, Golgi complexes, mitochondria, multivesicular bodies, and coated vesicles greatly increased in amounts. Nuclei displayed less heterochromatin and contained prominent nucleoli. These morphological changes were associated with transition from the free-living to the parasitic mode in life. The results were correlated with the fact that the hypodermis in N. brasiliensis is actively synthesizing culticular collagen during the third molt.This investigation was supported, in part, by awards 1-F02-AI3750-02 and AI-09625 from the National Institutes of Health, United States Public Health Service.We are grateful to Lida Petruniak and Dora Lou for technical assistance.     

Tracheal carbohydrate antigens identified by monoclonal antibodies

In a previous study we described a family of monoclonal antibodies directed against tracheal antigens having a variety of cellular and subcellular distributions. In the present study, we have extended our findings on four representative antibodies to determine the periodate sensitivity, glycosidase sensitivity, and apparent molecular weight of the corresponding antigens. Since mild periodate oxidation selectively cleaves carbohydrate moiety leaving amino acids intact, loss of antigenicity following this treatment suggests the involvement of sugar residues in the antigenic determinant. This can be confirmed by testing the sensitivity of the antigens to specific glycosidases. By enzyme-linked immunosorbent assay (ELISA), all four antibodies were found to have highest affinity for void volume components isolated by Bio-Gel A15m chromatography of the total tracheal secretion. Further analysis of this void volume material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions followed by immunoblot analysis revealed that all antigens were carried by high-molecular-weight species (greater than 200,000) which were periodate-Schiff positive but reacted poorly with Coomassie blue. In parallel experiments using immunofluorescence and ELISA, antibody binding was compared under control conditions and following periodate treatment of antigens under varying intensities (10 mM IO4-, 10 min, 4 degrees C; 50 mM IO4-, 1 h, 4 degrees C; 100 mM IO4-, 12 h, 20 degrees C). Similar results were obtained with the two methods, indicating a partial loss of antigenicity for one of the four antigens following the mildest periodate treatment, and total loss of antigenicity for all four antigens following each of the two prolonged treatments. All four antigens showed marked sensitivity to digestion with mixed exoglycosidases and three antigens were also susceptible to endo-beta-galactosidase digestion. Antigenicity was not decreased during incubation with chondroitinase ABC, heparitinase, or heparinase. Immunofluorescence analysis of tracheal tissue sections showed that the four antibodies recognized determinants in different locations, including gland and goblet cell cytoplasmic granules and the apical epithelial membrane. The characteristic immunofluorescence patterns of all antibodies were abolished by periodate incubation of the tracheal sections. Thus, the four antibodies appear to recognize carbohydrate antigens carried by high-molecular-weight glycoproteins, each with different cellular origins.