Elongated TCR alpha chain CDR3 favors an altered CD4 cytokine profile.

BACKGROUND: CD4 T lymphocyte activation requires T cell receptor (TCR) engagement by peptide/MHC (major histocompatibility complex) (pMHC). The TCR complementarity-determining region 3 (CDR3) contains variable α and ß loops critical for pMHC recognition. During any immune response, tuning of TCR usage through progressive clonal selection occurs. Th1 and Th2 cells operate at different avidities for activation and display distinct transcriptional programs, although polarization may be plastic, influenced by pathogens and cytokines. We therefore hypothesized that CDR3αß sequence features may intrinsically influence CD4 phenotype during progression of a response. RESULTS: We show that CD4 polarization involves distinct CDR3α usage: Th1 and Th17 cells favored short TCR CDR3α sequences of 12 and 11 amino acids, respectively, while Th2 cells favored elongated CDR3α loops of 14 amino acids, with lower predicted affinity. The dominant Th2- and Th1-derived TCRα sequences with 14 amino acid CDR3 loops and 12 amino acid CDR3 loops, respectively, were expressed in TCR transgenics. The functional impact of these TCRα transgenes was assessed after in vivo priming with a peptide/adjuvant. The short, Th1-derived receptor transgenic T cell lines made IFNγ, but not IL-4, 5 or 13, while the elongated, Th2-derived receptor transgenic T cell lines made little or no IFNγ, but increased IL-4, 5 and 13 with progressive re-stimulations, mirrored by GATA-3 up-regulation. T cells from primed Th2 TCRα transgenics selected dominant TCR Vß expansions, allowing us to generate TCRαß transgenics carrying the favored, Th2-derived receptor heterodimer. Primed T cells from TCRαß transgenics made little or no IL-17 or IFNγ, but favored IL-9 after priming with Complete Freund's adjuvant and IL-4, 5, 9, 10 and 13 after priming with incomplete Freund's. In tetramer-binding studies, this transgenic receptor showed low binding avidity for pMHC and polarized T cell lines show TCR avidity for Th17 > Th1 > Th2. While transgenic expression of a Th2-derived, 'elongated' TCR-CDR3α and the TCRαß pair, clearly generated a program shifted away from Th1 immunity and with low binding avidity, cytokine-skewing could be over-ridden by altering peptide challenge dose. CONCLUSION: We propose that selection from responding clones with distinctive TCRs on the basis of functional avidity can direct a preference away from Th1 effector responses, favoring Th2 cytokines.

Cancrincolidae (Copepoda, Harpacticoida) associated with land crabs: a semiterrestrial leaf of the ameirid tree.

Morphological evidence suggests harpacticoid copepods have recurrently entered into symbiosis with other crustaceans but only members of the family Cancrincolidae have successfully made the transition from marine habitats to semiterrestrial hosts. Cancrincolids are primarily amphi-Atlantic in distribution (with one outlier in the western Pacific) and typically inhabit the gill chambers of grapsoidean land crabs belonging to the families Grapsidae, Sesarmidae, Varunidae and Gecarcinidae. Morphologically, they are difficult to place because they exhibit unusual autapomorphies and the shared derived characters claimed to unite them with the primitively marine Ameiridae are equivocal. Both maximum parsimony and Bayesian inference solutions based on SSU rDNA gene sequences show topological congruence in placing the Cancrincolidae within the Ameiridae and in firmly resolving it as the sistergroup of taxa that have been reported as obligate or commensal associates of crayfish. This relationship is further supported by swimming leg sexual dimorphism and mandibular palp morphology. Morphological comparison with ameirid copepods revealed the majority of synapomorphies previously proposed in support of cancrincolid monophyly and familial distinctiveness can be attributed to heterochrony. The progressive evolution of cancrincolid associates appears to be largely concordant with the sequential adaptation to terrestriality by their grapsoidean hosts. The current amphi-Atlantic distribution of Cancrincola may suggest its possibly free-living ancestor had already assumed a virtually continuous distribution along the northern seaboard of Gondwana prior to the opening of the South Atlantic during the early Cretaceous, implying symbiotic relationships were established only much later when grapsoidean crabs started to emerge, radiate and diversify in the mid-Tertiary (15-35 mya). The adoption of semi-terrestriality in cancrincolid copepods can be viewed as yet another independent attempt (incursion) to colonize low-salinity environments by members of the family Ameiridae. The possible sistergroup relationships of the Ameiridae and the position of the genus Argestigens Willey are discussed.

Evolution of the bomolochiform superfamily complex (Copepoda: Cyclopoida): new insights from ssrDNA and morphology, and origin of Umazuracolids from polychaete-infesting ancestors rejected.

Poecilostome cyclopoids are among the most morphologically diverse copepods, having established symbiotic relationships with teleosts, elasmobranchs and invertebrate hosts belonging to no fewer than 14 marine phyla. Many parasitic lineages display radically divergent body plans and on that basis have traditionally been placed at higher taxonomic rank than they deserve. The most recent example is the monotypic family Umazuracolidae, established for a derived fish parasite with bomolochiform affinities. Phylogenetic analysis of complete ssrDNA (18S) sequences of 44 species belonging to 21 families of cyclopoid copepods shows that there is no support for the familial distinctiveness of the Umazuracolidae. Both maximum parsimony tree reconstruction and Bayesian inference, operating under the GTR+I+Γ model of nucleotide substitution, unambiguously placed Umazuracola elongatus in the Taeniacanthidae within the predominantly fish parasitic bomolochiform complex, refuting the original suggestion of a shared most recent common ancestry with polychaete symbionts. The phylogenies also revealed that the bomolochiform families and the Clausidiidae (and allies) form a monophyletic group, the clausidiiform complex, with high nodal support under both methods. Bayesian inference suggested a diphyletic origin of the "Poecilostomatoida" with the clausidiiform family-group holding a basal position while the traditional cyclopoid families form a monophyletic group in apposition to a second poecilostomatoid clade; however, maximum parsimony results were equivocal, depending on outgroup selection. Scrutiny of the morphological characters diagnosing the monotypic families Tegobomolochidae and Tuccidae demonstrated that they merely represent derived lineages within more inclusive taxa, the former being related to a group of nostril-inhabiting genera within the Bomolochidae, the latter forming the sistergroup of Taeniacanthodes within the Taeniacanthidae. The taeniacanthid genus Makrostrotos occupies a position at the base of the bomolochiform complex and is fixed as the type of a new family, Makrostrotidae. Although both morphological and molecular evidence hint that the Bomolochidae is nested within a paraphyletic Taeniacanthidae, the status quo of maintaining both families is favoured here pending additional molecular data. The bomolochiform complex, comprising the Bomolochidae, Taeniacanthidae, Telsidae and Makrostrotidae, is attributed superfamilial rank as the Bomolochoidea. A recent controversial phylogenetic analysis of the poecilostomatoid families is shown to be flawed, being based on a dataset containing imperfect or misleading information, and characters whose states were wrongly assessed.

Genetic diversity of Ascaris in southwestern Uganda.

Despite the common occurrence of ascariasis in southwestern Uganda, helminth control in the region has been limited. To gain further insights into the genetic diversity of Ascaris in this area, a parasitological survey in mothers (n=41) and children (n=74) living in two villages, Habutobere and Musezero, was carried out. Adult Ascaris worms were collected from infected individuals by chemo-expulsion using pyrantel pamoate treatment. Genetic diversity within these worms was assessed by inspection of DNA sequence variation in a mitochondrial marker and length polymorphism at microsatellite loci. Overall prevalence of ascariasis was 42.5% in mothers and 30.4% in their children and a total of 98 worms was examined from 18 hosts. Sequence analysis of a portion of the mitochondrial cytochrome c oxidase subunit 1 gene revealed 19 different haplotypes, 13 of which had not been previously encountered. Microsatellite analysis using eight loci provided evidence for high gene flow between worm populations from the two villages but comparing these worms with others obtained in a prior study on Unguja, Zanzibar, confirmed little genetic exchange and mixing of worm populations between the two areas. By adding to our understanding of the genetic diversity of Ascaris in Africa, this study provides useful information for monitoring changes in parasite population structure in the face of ongoing and future control.

Extraordinary host switching in siphonostomatoid copepods and the demise of the Monstrilloida: integrating molecular data, ontogeny and antennulary morphology.

Copepods exhibit an astounding variety of lifestyles, host associations and morphology, to the extent that their crustacean affinities may be obscured. Relationships among the ten copepod orders based on morphological characters remain equivocal. Here we test the ordinal status of the enigmatic Monstrilloida using SSU rDNA gene sequences, comparative morphological data (antennulary sensory interface) and ontogenetic data (caudal ramus setation patterns). Bayesian analysis unexpectedly revealed the Monstrilloida are nested within a fish-parasitic clade of the Siphonostomatoida and share a common ancestor with the stem species of the caligiform families (sea-lice). This unforeseen relationship is congruent with both antennulary and caudal ramus morphology. The divergence of the monstrilloids from an ectoparasitic, vertebrate-associated ancestor involved radical changes in host utilization, body plan and life cycle strategy, a combination rarely observed and probably unique in metazoan parasites. Adult monstrilloids secondarily returned to a free-living, predator-exposed mode of life and we postulate the pressure on maintaining a functional approaching-predator detection system has progenetically delayed the suppression (as in post-copepodid caligiform instars) of the 5-point antennulary sensory array. The homoplastic evolution of the frontal filament in Siphonostomatoida is discussed.