Differential proliferative response of NKT cell subpopulations to in vitro stimulation in presence of different cytokines

Human Valpha24(+)Vbeta11(+) NKT (NKT) cells have immune regulatory activities associated with rejection of tumors, infections and control of autoimmune diseases. They can be stimulated to proliferate using alpha-galactosylceramide (KRN7000) and have the potential for therapeutic manipulation. Subpopulations of NKT cells (CD4(+)CD8(-), CD4(-)D8(+) and CD4(-)CD8(-)) have functionally distinctive Th1/Th2 cytokine profiles and their relative numbers following stimulation may influence the Th1/Th2 balance, which may result in or prevent disease. We aimed to determine the effect of different cytokines in culture during stimulation of NKT cells on the relative proportions of NKT cell subpopulations. Our results show that all NKT cell subpopulations expanded following stimulation with KRN7000 and IL-2, IL-7, IL-1 2 or IL-15. Expansion capacity differed between subpopulations, resulting in different relative proportions of CD4(+) and CD4(-) NKT cell subpopulations, and this was influenced by the cytokine used for stimulation. A Th1-biased environment was observed after stimulation of NKT cells. NKT cells expanded under all conditions evaluated demonstrated significant cytotoxicity against U937 tumor cells. In view of the potential for NKT cell subsets to alter the balance of Th1 and Th2 environment, these data provide insights into the effects of NKT cell manipulation for possible therapeutic applications in different disease settings.

Observations of spermiogenesis and epididymal sperm maturation in the rufous hare wallaby, Lagorchestes hirsutus (Metatheria, Mammalia)

Acrosomal development in the early spermatid of the rufous hare wallaby shows evidence of formation of an acrosomal granule, similar to that found in eutherian mammals, the Phascolarctidae and Vombatidae. Unlike the other members of the Macropodidae so far examined, the acrosome of this species appears to be fully compacted at spermiation and extends evenly over 90% of the dorsal aspect of the nucleus. During spermiogenesis, the nucleus of the rufous hare wallaby spermatid showed evidence of uneven condensation of chromatin; this may also be related to the appearance of unusual nucleoplasm evaginations from the surface of the fully condensed spermatid. This study was unable to find evidence of the presence of Sertoli cell spurs or nuclear rotation during spermiogenesis in the rufous hare wallaby. The majority of spermatozoa immediately before spermiation had a nucleus that was essentially perpendicular to the long axis of the sperm tail. Nuclei of spermatozoa found in the process of being released or isolated in the lumen of the seminiferous tubule were rotated almost parallel to the long axis of the flagellum; complete parallel alignment occurred during epididymal maturation. At spermiation spermatozoa have characteristically small cytoplasmic remnants compared to those of other macropods. Unlike the majority of macropodid spermatozoa so far described, the spermatozoa of the rufous hare wallaby showed little evidence of morphological change during epididymal transit. There was no formation of a fibre network around the midpiece or of plasma membrane specializations in this region; the only notable change was a distinctive flattening of midpiece mitochondria and scalloping of the anterior mitochondrial sheath to accommodate the sperm head. Preliminary evidence from spermiogenesis and epididymal sperm maturation supports the classification of the rufous hare wallaby as a separate genus but also indicates that its higher taxonomic position may need to be re-evaluated.

Sperm membrane fatty acid composition in the Eastern grey kangaroo (Macropus giganteus), koala (Phascolarctos cinereus), and common wombat (Vombatus ursinus) and its relationship to cold shock injury and cryopreservation success

Marsupial spermatozoa tolerate cold shock well, but differ in cryopreservation tolerance. In an attempt to explain these phenomena, the fatty acid composition of the sperm membrane from caput and cauda epididymides of the Eastern grey kangaroo, koala, and common wombat was measured and membrane sterol levels were measured in cauda epididymidal spermatozoa. While species-related differences in the levels of linolenic acid (18:3, n-6) and arachidonic acid (20:4, n-6) were observed in caput epididymal spermatozoa, these differences failed to significantly alter the ratio of unsaturated/saturated membrane fatty acids. However in cauda epididymidal spermatozoa, the ratio of unsaturated/saturated membrane fatty acids in koala and kangaroo spermatozoa was approximately 7.6 and 5.2, respectively; substantially higher than any other mammalian species so far described. Koala spermatozoal membranes had a higher ratio of unsaturated/saturated membrane fatty acids than that of wombat spermatozoa (t = 3.81; df = 4; p less than or equal to 0.02); however, there was no significant difference between wombat and kangaroo spermatozoa. The highest proportions of DHA (22:6, n-3), the predominant membrane fatty acid in cauda epididymidal spermatozoa, were found in wombat and koala spermatozoa. While species-related differences in membrane sterol levels (cholesterol and desmosterol) were observed in cauda epididymidal spermatozoa, marsupial membrane sterol levels are very low. Marsupial spermatozoal membrane analyses do not support the hypothesis that a high ratio of saturated/unsaturated membrane fatty acids and low membrane sterol levels predisposes spermatozoa to cold shock damage. Instead, cryogenic tolerance appears related to DHA levels. (C) 2004 Elsevier Inc. All rights reserved.

Sperm ultrastructure of the Actinocyclidae (Mollusca, Nudibranchia) and homology of the terminal region of nudibranch sperm

Sperm ultrastructure is examined and described for the actinocyclidid nudibranchs Actinocyclus verrucosus, Hallaxa iju and Hallaxa indecora. Although general characteristics were consistent with previously described heterobranch observations, present investigations revealed ultrastructural synapomorphies for the family based on the morphology of the terminal region of the spermatozoon. In actinocyclidids, the axonemal microtubules penetrate for some distance beyond the annulus, and the annular accessory body elongates to completely seal the terminal region. Chromodoris also has an annular accessory body that completely seals the axoneme and terminal region, but it does not extend far beyond the annulus, and it is possible that these states were derived independently. Cytochemical staining confirmed that there was no glycogen present in the posterior region of the sperm for H. indecora or Chromodoris kuniei. However, representatives of other chromodoridid genera (Noumea, Risbecia) have an axoneme that penetrates through the entire annular complex, after which it is sheathed by a glycogen deposit. Similarities in the acrosomal complex support the proposed sister group relationship between the Actinocyclidae and Chromodorididae.

Analysis of the effect of different NKT cell subpopulations on the activation of CD4 and CD8 T cells, NK cells, and B cells

Objective. NKT cells have diverse immune regulatory functions including activation of cells involved in Th1- and Th2-type immune activities. Most previous studies have investigated the functions of NKT cells as a single family but more recent evidence indicates the distinct functional properties of NKT cell subpopulation. This study aims to determine whether NKT cell subpopulations have different stimulatory activities on other immune cells that may affect the outcome of NKT cell-based immunotherapy. Methods. NKT cells and NKT cell subpopulations (CD4(+)CD8(-), CD4(-)CD8(+), CD4(-)CD8(+)) were cocultured with PBMC and their activities on immune cells including CD4(+) and CD8(+) T cells, NK cells, and B cells were assessed by flow cytometry. The production of cytokines in culture was measured by enzyme-linked immunsorbent assay. Results. The CD4(+)CD8(-) NKT cells demonstrated substantially greater stimulatory activities on CD4(+) T cells, NK cells, and B cells than other NKT cell subsets. The CD4(-)CD8(+) NKT cells showed the greatest activity on CD8(+) T cells, and were the only NKT cell subset that activated these immune cells. The CD4(-)CD8(-) NKT cells showed moderate stimulatory activity on CD4(+) T cells and the least activity on other immune cells. Conclusion. The results here suggest that NKT cell subpopulations differ in their abilities to stimulate other immune cells. This highlights the potential importance of manipulating specific NKT cell subpopulations for particular therapeutic situations and of evaluating subpopulations, rather than NKT cells as a group, during investigation of a possible role of NKT cells in various disease settings. (c) 2006 International Society for Experimental Hematology. Published by Elsevier Inc.

Basal chromodorid sperm ultrastructure (Nudibranchia, Gastropoda, Mollusca)

The relationship between three genera considered basal in the Chromodorididae (Cadlina, Tyrinna, Cadlinella) has not yet been resolved by traditional morphological means. Here we examined the sperm ultrastructure of Tyrinna nobilis, Tyrinna evelinae, Cadlina flavomaculata and Cadlina cf. nigrobranchiata, with the expectation of finding phylogenetically informative characters. No Tyrinna or Cadlina species showed sperm similarities to Cadlinella. Both Cadlina species and Tyrinna nobilis (but not T. evelinae) exhibited coarse striations in the acrosomal pedestal. The putative fibers that occurred between the coarse striations of the pedestal are condensed into a layer in Cadlina and Tyrinna, but not in other species that also have coarse striations (Gymnodoris), and may constitute evidence for a close relationship. Tyrinna evelinae possessed fine acrosomal striations, which was shared with other Chromodorididae, Actinocyclidae and the cryptobranchs Rostanga and Aphelodoris. We also examined the sperm ultrastructure of 'Chromodoris' ambiguus, an animal which has shown molecular affinities to species of Cadlina, and not Chromodoris. The sperm of 'C'.' ambiguus did not exhibit the typical Cadlina characteristics, but also showed important differences to other investigated Chromodoris species.

Comparative gene expression analysis of NKT cell subpopulations

Natural killer T (NKT) cells are a lymphocyte lineage, which has diverse immune regulatory activities in many disease settings. Most previous studies have investigated the functions of this family of cells as a single entity, but more recent evidence highlights the distinct functional and phenotypic properties of NKT cell subpopulations. It is likely that the diverse functions of NKT cells are regulated and coordinated by these different NKT subsets. Little is known about how NKT subsets differ in their interactions with the host. We have undertaken the first microarray analysis comparing the gene expression profiles of activated human NKT cell subpopulations, including CD8(+) NKT cells, which have often been overlooked. We describe the significant gene expression differences among NKT cell subpopulations and some of the molecules likely to confer their distinct functional roles. Several genes not associated previously with NKT cells were shown to be expressed differentially in specific NKT cell subpopulations. Our findings provide new insights into the NKT cell family, which may direct further research toward better manipulation of NKT cells for therapeutic applications.

One-sided ejaculation of sperm bundles in the echidna

We report for the first time an unusual ejaculatory mechanism in the short-beaked echidna in which each side of the bilaterally symmetrical, rosettelike glans penis is used alternately, with the other being shut down. This is unparalleled in mammals but is reminiscent of the use of hemipenes in squamate reptiles, providing further reproductive evidence of a sauropsidian lineage in the Monotremata. Further, we describe the occurrence of motile sperm bundles in ejaculated echidna semen and provide scanning electron micrographs of their morphology. Sperm bundling appears to confer increased sperm motility, which may provide the potential for sperm competition between males.