Regulatory T cells suppress CD8+ T cell responses induced by direct priming and cross-priming and moderate immunodominance disparities

Little is known regarding the participation of CD4+ CD25+ regulatory T cells (Treg) in TCD8+ responses. In this study, we show that Treg depletion via treatment with anti-CD25 mAb (PC61) significantly enhances TCD8+ responses to influenza A virus, vaccinia virus, and SV40-transformed cells induced by either direct priming or cross-priming. PC61 did not enhance TCD8+ responses in CD4-deficient mice, providing the initial demonstration that PC61 acts on a subset of TCD4+, and not on other cells that express either CD25 or a fortuitously cross-reactive Ag. We further show that Treg selectively suppress responses to the most immunodominant TCD8+ determinants in the three systems examined. Therefore, Treg influence TCD8 immunodominance hierarchies by moderating disparities in responses to different determinants.     

Trypanosoma cruzi contains a single detectable uracil-DNA glycosylase and repairs uracil exclusively via short patch base excision repair

Enzymes involved in genomic maintenance of human parasites are attractive targets for parasite-specific drugs. The parasitic protozoan Trypanosoma cruzi contains at least two enzymes involved in the protection against potentially mutagenic uracil, a deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and a uracil-DNA glycosylase belonging to the highly conserved UNG-family. Uracil-DNA glycosylase activities excise uracil from DNA and initiate a multistep base-excision repair (BER) pathway to restore the correct nucleotide sequence. Here we report the biochemical characterisation of T.cruzi UNG (TcUNG) and its contribution to the total uracil repair activity in T.cruzi. TcUNG is shown to be the major uracil-DNA glycosylase in T.cruzi. The purified recombinant TcUNG exhibits substrate preference for removal of uracil in the order ssU>U:G>U:A, and has no associated thymine-DNA glycosylase activity. T.cruzi apparently repairs U:G DNA substrate exclusively via short-patch BER, but the DNA polymerase involved surprisingly displays a vertebrate POLdelta-like pattern of inhibition. Back-up UDG activities such as SMUG, TDG and MBD4 were not found, underlying the importance of the TcUNG enzyme in protection against uracil in DNA and as a potential target for drug therapy.     

Fasciolagigantica: Parasitological and scanning electron microscopy study of the in vitro effects of ivermectin and/or artemether

Objective

To evaluate the in vitro effects of different concentrations of ivermectin and/or artemether on Fasciolagigantica worms and to study the parasitological changes and tegumental alterations using scanning electron microscopy (SEM).

Methods

Fasciola gigantica worms were incubated in vitro for 24 and 48 h with three concentrations of either ivermectin or artemether (10, 20 and 50 μg/ml) or both in half concentration of either (5, 10 and 25 μg/ml).

Results

Exposure of Fasciola worms to 25 + 25 μg/ml of combined drug regimens or to 50 μg/ml of either ivermectin or artemether for 48 h led to 100%, 41.7% and 75% worm killing which were accompanied by a significant reduction in egg laying capacity and significant increase in dead eggs maximally recorded in combined drug regimens. SEM of the flukes incubated for 48 h with combined drug regimens showed maximal tegumental disruption with swelling of the worm body, roughness, blebbing, sloughing and complete loss of spines. Disruption to the tegument of the flukes induced by artemether was more than that of ivermectin.

Conclusions

Artemether alone or combined with ivermectin in half doses had potent fasciocidal activities. Besides, half doses of combined drug regimens had higher ovicidal effects than each drug alone. In vivo studies are recommended to explore the efficacy of combined regimens against Fasciola infection.     

Hybrids in divers (Gaviiformes)

The incidence of hybridisation in birds differs greatly between orders and is expected to be low in orders comprising few species. The divers or loons (Gaviiformes) are a species-poor group in which apparent hybridisation has been reported infrequently. Here we report on a hybrid diver stored in the collections of the Zoological Museum Amsterdam. The bird shows a heterogeneous set of characters, some shared with the putative parent species White-billed Diver Gavia adamsii and Great Northern Diver G. immer, others being intermediate between the two. A Canonical Discriminant Function analysis positions the bird between these two putative parent species, making a hybrid status quite likely. We evaluate the evidence for hybridisation in the order Gaviiformes and conclude that hybridisation has been suspected in four of the five species, though documentation is limited. If this high incidence could be confirmed, it would rank among the highest of any avian order, contradicting the assumption that incidence of hybridisation in small orders is relatively low.     

Preventing and curing citrulline-induced autoimmune arthritis in a humanized mouse model using a Th2-polarizing iNKT cell agonist

Invariant natural killer T (iNKT) cells are innate lymphocytes with unique reactivity to glycolipid antigens bound to non-polymorphic CD1d molecules. They are capable of rapidly releasing pro- and/or anti-inflammatory cytokines and constitute attractive targets for immunotherapy of a wide range of diseases including autoimmune disorders. In this study, we have explored the beneficial effects of OCH, a Th2-polarizing glycolipid agonist of iNKT cells, in a humanized mouse model of rheumatoid arthritis (RA) in which citrullinated human proteins are targeted by autoaggressive immune responses in mice expressing an RA susceptibility human leukocyte antigen (HLA) DR4 molecule. We found for the first time that treatment with OCH both prevents and cures citrulline-induced autoimmune arthritis as evidenced by resolved ankle swelling and reversed histopathological changes associated with arthritis. Also importantly, OCH treatment blocked the arthritogenic capacity of citrullinated antigen-experienced splenocytes without compromising their global responsiveness or altering the proportion of splenic naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T cells. Interestingly, administering the Th1-promoting iNKT cell glycolipid ligand α-C-galactosylceramide into HLA-DR4 transgenic mice increased the incidence of arthritis in these animals and exacerbated their clinical symptoms, strongly suggesting a role for Th1 responses in the pathogenesis of citrulline-induced arthritis. Therefore, our findings indicate a role for Th1-mediated immunopathology in citrulline-induced arthritis and provide the first evidence that iNKT cell manipulation by Th2-skewing glycolipids may be of therapeutic value in this clinically relevant model, a finding that is potentially translatable to human RA.     

Statin therapy lowers muscle sympathetic nerve activity and oxidative stress in patients with heart failure

Despite standard drug therapy, sympathetic nerve activity (SNA) remains high in heart failure (HF) patients making the sympathetic nervous system a primary drug target in the treatment of HF. Studies in rabbits with pacing-induced HF have demonstrated that statins reduce resting SNA, in part, due to reductions in reactive oxygen species (ROS). Whether these findings can be extended to the clinical setting of human HF remains unclear. We first performed a study in seven statin-na?ve HF patients (56 ± 2 yr; ejection fraction: 31 ± 4%) to determine if 1 mo of simvastatin (40 mg/day) reduces muscle SNA (MSNA). Next, to control for possible placebo effects and determine the effect of simvastatin on ROS, a double-blinded, placebo-controlled crossover design study was performed in six additional HF patients (51 ± 3 yr; ejection fraction: 22 ± 4%), and MSNA, ROS, and superoxide were measured. We tested the hypothesis that statin therapy decreases resting MSNA in HF patients and this would be associated with reductions in ROS. In study 1, simvastatin reduced resting MSNA (75 ± 5 baseline vs. 65 ± 5 statin bursts/100 heartbeats; P < 0.05). Likewise, in study 2, simvastatin also decreased resting MSNA (59 ± 5 placebo vs. 45 ± 6 statin bursts/100 heartbeats; P < 0.05). In addition, statin therapy significantly reduced total ROS and superoxide. As expected, cholesterol was reduced after simvastatin. Collectively, these findings indicate that short-term statin therapy concomitantly reduces resting MSNA and total ROS and superoxide in HF patients. Thus, in addition to lowering cholesterol, statins may also be beneficial in reducing sympathetic overactivity and oxidative stress in HF patients.     

Productive replication and evolution of HIV-1 in ferret cells

A rodent or other small animal model for HIV-1 has not been forthcoming, with the principal obstacles being species-specific restriction mechanisms and deficits in HIV-1 dependency factors. Some Carnivorans may harbor comparatively fewer impediments. For example, in contrast to mice, the domestic cat genome encodes essential nonreceptor HIV-1 dependency factors. All Feliformia species and at least one Caniformia species also lack a major lentiviral restriction mechanism (TRIM5α/TRIMCyp proteins). Here we investigated cells from two species in another carnivore family, the Mustelidae, for permissiveness to the HIV-1 life cycle. Mustela putorius furo (domesticated ferret) primary cells and cell lines did not restrict HIV-1, feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), or N-tropic murine leukemia virus (MLV) postentry and supported late HIV-1 life cycle steps comparably to human cells. The ferret TRIM5α gene exon 8, which encodes the B30.2 domain, was found to be pseudogenized. Strikingly, ferret (but not mink) cells engineered to express human HIV-1 entry receptors supported productive spreading replication, amplification, and serial passage of wild-type HIV-1. Nevertheless, produced virions had relatively reduced infectivity and the virus accrued G→A hypermutations, consistent with APOBEC3 protein pressure. Ferret cell-passaged HIV-1 also evolved amino acid changes in the capsid cyclophilin A binding loop. We conclude that the genome of this carnivore can provide essential nonreceptor HIV-1 dependency factors and that ferret APOBEC3 proteins with activity against HIV-1 are likely. Even so, unlike in cat cells, HIV-1 can replicate in ferret cells without vif substitution. The virus evolves in this novel nonprimate cell adaptive landscape. We suggest that further characterization of HIV-1 adaptation in ferret cells and delineation of Mustelidae restriction factor repertoires are warranted, with a view to the potential for an HIV-1 animal model.     

Heterologous virus-induced gene silencing as a promising approach in plant functional genomics

VIGS (virus induced gene silencing) is considered as a powerful genomics tool for characterizing the function of genes in a few closely related plant species. The investigations have been carried out mainly in order to test if a pre-existing VIGS vector can serve as an efficient tool for gene silencing in a diverse array of plant species. Another route of investigation has been the constructing of new viral vectors to act in their hosts. Our approach was the creation of a heterologous system in which silencing of endogenous genes was achieved by sequences isolated from evolutionary remote species. In this study, we showed that a TRV-based vector cloned with sequences from a gymnosperm, Taxus baccata L. silenced the endogenous phytoene desaturase in an angiosperm, N. benthamiana. Our results showed that inserts of between 390 and 724 bp isolated from a conserved fragment of the Taxus PDS led to silencing of its homolog in tobacco. The real time analysis indicated that the expression of PDS was reduced 2.1- to 4.0-fold in pTRV-TbPDS infected plants compared with buffer treated plants. Once the best insert is identified and the conditions are optimized for heterologous silencing by pTRV-TbPDS in tobacco, then we can test if TRV can serve as an efficient silencing vector in Taxus. This strategy could also be used to silence a diverse array of genes from a wide range of species which have no VIGS protocol. The results also showed that plants silenced heterologously by the VIGS system a minimally affected with respect to plant growth which may be ideal for studying the genes that their complete loss of function may lead to decrease of plant growth or plant death.