Large Particle Sorting to Isolate Live Parasitic Nematode Eggs.

Traditional jet-in-air cell sorters have been designed and optimized to isolate small particles such as mammalian lymphocytes with an average diameter of 10 µm. We discuss the practical considerations of setting up a conventional jet-in-air cell sorter, using a 200-µm nozzle, to isolate the large parasitic nematode eggs of Nippostrongylus brasiliensis, with a maximum size of 60 µm. The eggs were separated based on light scattering properties, no fluorescent dye or molecule was required.

The role of complement in innate, adaptive and eosinophil-dependent immunity to the nematode Nippostrongylus brasiliensis.

Complement may be important for immunity to infection with parasitic helminths, by promoting the recruitment of leukocytes to infected tissues and by modulating the function of cytotoxic effector leukocytes. However, the importance of complement in vivo during helminth infection is poorly understood. In this study, mice lacking classical (C1q-deficient), alternative (factor B-deficient) or all pathways of complement activation (C3-deficient) were used to assess the role of complement in immunity to the nematode Nippostrongylus brasiliensis. Double-mutant complement-deficient/IL-5 transgenic (Tg) mice were used to determine if complement is required for the strong eosinophil-dependent resistance to this parasite. Complement activation on larvae (C3 deposition), extracellular eosinophil peroxidase activity, larval aggregation and eosinophil recruitment to the skin 30 min post-injection (p.i.) of larvae were reduced in factor B-deficient mice. Inhibition of the C5a receptor with the antagonist PMX53 impaired eosinophil and neutrophil recruitment to the skin. C3 deposition on larvae was minimal by 150 min p.i. and at this time cell adherence, larval aggregation, eosinophil recruitment and degranulation were complement-independent. Factor B and C3 deficiency were associated with higher lung larval burdens in primary infections. Complement-deficient/IL-5 Tg mice were highly resistant to N. brasiliensis, suggesting that eosinophils can limit infection in a complement-independent manner. Potent secondary immunity was similarly complement-independent. In conclusion, although the alternative pathway is important for parasite recognition and leukocyte recruitment early in N. brasiliensis infections, the parasite soon becomes resistant to complement and other factors can compensate to promote eosinophil-dependent immunity.

Morphological differentiation and function of the coelomocytes in the parasitic stages of Nippostrongylus brasiliensis.

Female and male worms of Nippostrongylus brasiliensis exhibited sexual dimorphism based on the number of coelomocytes present. A surprising multiplicity of diverse morphological types of coelomocytes developed in both female and male worms during the parasitic cycle. Cytoplasmic processes began to appear on the surface membrane of coelomocytes in the late third-stage larvae (L3s) in the lungs, and they increased greatly in type, size, and morphology during the fourth and fifth stages. These structures were characterized primarily as complex filopodia, pseudopodia, and cytoplasmic pearls, which resulted in the formation of highly pleomorphic cells. Pearls, starting as small protuberances, progressively increased in size and number with larval growth and development. In the adult worms, a novel process of autocannibalism was initiated in many of the very large coelomocytes. The pearls grew enormously in size at the expense of the cytoplasm, forming a peripheral garland in 1 plane surrounding a residual, small, flat, cytoplasmic core containing the nucleus. The underlying "strategy" was to increase the surface-to-volume ratio of these huge cells to overcome the restriction imposed by eutely; the coelomocytes do not undergo cell division. This morphological innovation makes possible a more efficient uptake of nutrients and exocytosis of waste matter. Vesicles (presumably lysosomes) in the coelomocytes of the infective L3 store an extraordinarily high concentration of vitamin B12 (cobalamin, Cbl). At present, the only physiological function that can be assigned to coelomocytes of N. brasiliensis is the uptake, concentration, and storage of Cbl in the free-living stages, with the subsequent release of the molecule from the vesicles in the early phase of parasitism. Thus, stored Cbl in the infective L3 is made available for biochemical processes during the critical period of larval growth and differentiation initiated in the lung. A model of a hypothetical coelomocyte is presented relative to the processing and use of Cbl. Based on many criteria, it is possible that functional differences exist between different morphological types of coelomocytes in the parasitic stages of N. brasiliensis and that future studies will have to address this matter.

The origin of coelomocytes 5 and 6 and their interaction with a seam nurse cell in the free-living stages of Nippostrongylus brasiliensis.

A progenitor blast cell in the posterior of the newly hatched larva of Nippostrongylus brasiliensis yielded a large population of coelomic cells among which were 2 previously undiscovered coelomocytes, C5 and C6. The coelomocytes lay subdorsal and posterior to the genital primordium, C5 on the right and C6 on the left. Also, in the first-stage larva, 7 single seam cells appeared in the hypodermis on both the right and left sides arrayed in tandem along its length. Each seam cell (1-5, 7) went through 2 divisions with spaces maintained between the formed quartets. However, seam cell 6 underwent an unusual series of divisions resulting in the formation of a huge amoeboid nurse cell that enclosed a quartet of small cells in a vacuole; the quartet also was derived from seam cell 6. Ultimately, all the seam cells, including the nurse cells on each side of the larva, regressed and disappeared, except for the quartet cells, now released from their vacuole. These latter cells then remained dormant during the life of the free-living stages. During the process of seam cell development, coelomocytes 5 and 6 aligned themselves closely to seam cells 6 and their progeny; some attached themselves to and even partially penetrated the nurse cells at the level of the vacuole. At the time of the second molt and the formation of the early-third stage infective larva, tiny vesicles began to appear in the cytoplasm of coelomocytes 5 and 6. As vesicles increased in number, they aggregated into a mass at either the anterior or posterior pole of the cells. Coelomocytes 1-4 situated anterior to the genital primordium differed from coelomocytes 5 and 6 in that they accumulated much larger numbers of vesicles that remained discrete in the cytoplasm and concentrated extraordinary amounts of vitamin B12 that was recognized as a red pigment filling the vesicles. However, no red pigment ever was seen in the vesicles of coelomocytes 5 and 6. On the basis of the very early sexual differentiation of larvae in the rat lung, it was determined that the infective free-living larvae from which they were derived, and which contained coelomocytes 5 and 6, were female; those lacking coelomocytes 5 and 6 were presumed to be male.

Morphology and differentiation of the coelomocytes of the free-living stages of Nippostrongylus brasiliensis.

With the use of observations on living and fixed and stained larvae of Nippostrongylus brasiliensis reared in charcoal-feces cultures, an integrated view is presented on the morphology and differentiation of the coelomocytes present in the body cavity of the 2 rhabditiform stages and the exsheathed third stage. Four coelomocytes are present in the newly hatched larva arranged linearly from the base of the esophagus to the genital primordium. They lie subventrally, the anterior 2 on the right side of the ventral nerve cord, the posterior 2 on the left side. Data on the growth of these cells and analysis of their fixed site location in the coelom are presented. The coelomocytes are firmly attached to the inner surface of the body wall by filopodia. However, aberrantly positioned coelomocytes suggest the possibility that detachment and migration may occur. A few minute cytoplasmic inclusions are present in the coelomocytes in living newly hatched larvae and may reach approximately 100 or more in each coelomocyte in the third stage. These inclusions exhibit 2 phases of a pigmentation process. They are colorless in the rhabditiform stages, but simultaneous with the initiation of the second molt to form the infective larva, the inclusions rapidly turn a pink to rose hue. A variety of previous experimental data strongly support the interpretation that the pigment represents an extraordinary concentration of vitamin B12 in the coelomocytes obtained by larval feeding on bacterial B12 synthesizers present in the charcoal-feces cultures.