Long-term T cell memory to human leucocyte antigen-A2 supertype epitopes in humans vaccinated against smallpox.

Identification of human leucocyte antigen (HLA) class I-restricted T cell epitopes is important to develop methods to track the evolution of T cell memory to new generation smallpox vaccines and allow comparison to older vaccinia virus preparations known to induce protection against smallpox. We evaluated the relative predictive values of four computational algorithms to identify candidate 9-mer HLA-A2 supertype epitopes that were confirmed to stimulate preferentially T cell interferon (IFN)-gamma responses by subjects last vaccinated with Dryvax 27-54 years previously. Six peptides encoded by I4L, G1L, A8R, I8R, D12L and H3L open reading frames that were identical for Vaccinia (Copenhagen), Variola major (Bangledesh 1975) and modified vaccinia Ankara strain preferentially stimulated IFN-gamma responses by healthy HLA-A2 supertype adults last given Dryvax 27-49 years earlier relative to remotely vaccinated non-HLA-A2 supertype and unvaccinated HLA-A2 supertype adults. Combining results from at least two computational algorithms that use different strategies to predict peptide binding to HLA-A2 supertype molecules was optimal for selection of candidate peptides that were confirmed to be epitopes by recall of T cell IFN-gamma responses. These data will facilitate evaluation of the immunogenicity of replication incompetent smallpox vaccines such as modified vaccinia Ankara and contribute to knowledge of poxvirus epitopes that are associated with long-lived T cell memory.

Polymorphisms of innate immunity genes and susceptibility to lymphatic filariasis.

We examined 906 residents of an area of Papua New Guinea where bancroftian filariasis is endemic for genetic polymorphisms in three innate immunity genes suspected of contributing to susceptibility to infection and lymphatic pathology. Active infection was confirmed by the presence of blood-borne microfilariae and circulating filarial antigen in plasma. Disease was ascertained by physical examination for the presence of overt lymphedema (severe swelling of an arm or leg) or hydrocele. There was no association of infection status, lymphedema of an extremity, or hydrocele with chitotriosidase genotype (CHIT1). Polymorphisms of toll-like receptor-2 and toll-like receptor-4 genes (TLR4 A896G; TLR2 T2178A, G2258A) were not detected (N=200-625 individuals genotyped) except for two individuals heterozygous for a TLR2 mutation (C2029 T). These results indicate that a CHIT1 genotype associated previously with susceptibility to filariasis in residents of southern India and TLR2 and TLR4 polymorphisms do not correlate with infection status or disease phenotype in this Melanesian population.

Ecologic and biologic determinants of filarial antigenemia in bancroftian filariasis in Papua New Guinea.

The relationship between filarial antigenemia and lymphatic pathology was investigated in residents of 11 villages in an area of Papua New Guinea where Wuchereria bancrofti is endemic. Antigenemia was determined in 1322 persons by means of the Og4C3 antibody capture assay. Prevalence of antigenemia by village ranged from 61.7% to 98.2% and did not vary by sex. Antigen level increased with transmission potential among the 4 villages with measured transmission potential (r(2)=.945; P=.028). Antigenemia was associated positively with age in villages with the lowest annual transmission potentials (45 and 404 infective larvae/year; P<.001), but was distributed evenly across age groups in villages with increased transmission (1485 and 2518 infective larvae/year). These data suggest that children and adults have similar worm burdens in areas of high transmission, whereas worm burdens in areas of lower transmission increase with age. These results may be useful in the design and evaluation of programs aimed at eliminating lymphatic filariasis.

Activation of signaling pathways in HL60 cells and human neutrophils by farnesylthiosalicylate.

Effects of the farnesylcysteine mimetic, farnesylthiosalicylate on the activation of myeloid cells were studied. In dimethyl-sulfoxide-differentiated HL60 cells and in human neutrophils farnesylthiosalicylate (< or = 20 microM) dose-dependently elevated cytosolic Ca2+ concentrations, suggesting phospholipase-C-mediated release of the ion from intracellular stores. In human neutrophils, in addition to the production of inositol trisphosphate, farnesylthiosalicylate induced activation of the NADPH oxidase and translocation of the cytosolic oxidase components p47-phox and p67-phox to the membrane. The calcium signal, inositol-trisphosphate production and superoxide generation elicited by farnesylthiosalicylate were partially blocked by treatment of the cells with pertussis toxin, consistent with participation of pertussis-toxin-sensitive and pertussis-toxin-resistant elements. In HL60 cells, farnesylthiosalicylate (< or = 20 microM) did not activate NADPH oxidase but dose-dependently augmented PMA-elicited activity of the enzyme. This effect was resistant to pertussis-toxin treatment. In vitro augmentation of PKC-mediated phosphorylation of histone and cytosolic p47-phox by farnesylthiosalicylate and the finding that downregulation of PKC abrogated potentiation of NADPH oxidase activity by farnesylthiosalicylate were compatible with the involvement of PKC in the response of HL60 cells to farnesylthiosalicylate. It is suggested that the effects of farnesylthiosalicylate on myeloid cells reflect interaction of the analog with prenylcysteine-docking sites on cellular signaling elements.

The assembly of neutrophil NADPH oxidase: effects of mastoparan and its synthetic analogues.

Detergent-mediated activation of the phagocyte superoxide-generating NADPH oxidase requires the participation of at least four proteins: the membrane-bound heterodimeric cytochrome b558 and three cytosolic components, p47-phox, p67-phox and a Rac1/Rac2 protein. Peptides corresponding to sequences of different subunits of NADPH oxidase have been used as probes of the mechanism and sequence of assembly of the active complex. In the present study effects of mastoparans on activation of NADPH oxidase were investigated. Mastoparans are wasp venom cationic amphiphilic tetradecapeptides capable of modulation of various cellular activities. Natural mastoparans, as well as several synthetic mastoparan analogues, unrelated to oxidase components, blocked activation of the oxidase in the cell-free system (EC50 = 1.5 microM) and in guanosine 5'-[gamma-thio]triphosphate (GTP[S])/ATP-stimulated neutrophils permeabilized with streptolysin O. In the cell-free system the effect was not relieved by raising the detergent concentration and could not be ascribed to changes in critical micellar concentration values of the activating SDS or arachidonate. Chromatography of neutrophil cytosol on an immobilized mastoparan column suggested interaction of cytosolic p47-phox and p67-phox with the peptide. In spite of this interaction mastoparan did not interfere with translocation of p47-phox and p67-phox to the cell membranes.

Pulmonary bacterial deposition and clearance during ascarid larval migration in weanling pigs.

Pulmonary deposition and clearance of bacteria were measured in weanling pigs, half of which had been inoculated at age 31 days with larvated Ascaris suum ova. Seven days later, when breathing signs of larval migration were pronounced, all pigs were exposed to aerosolized Escherichia coli (strain B). Then, either immediately after aerosol exposure (for deposition assessment) or immediately after a 120 minute period in filtered air (for clearance), bacteria in the pigs' lungs were counted. Ascarid ova-inoculated pigs did not differ significantly from control pigs for number of bacteria in the lungs after aerosol exposure, but after the 120 minute clearance period they had 7.2 times more than did the control pigs. Thus, in weanling pigs, the breathing-pattern changes that were evident during ascarid-larval migration did not affect pulmonary deposition of inhaled bacteria significantly, but the presence of ascarid larvae in the lungs was associated with impaired pulmonary bacterial clearance.