NK cell immunophenotypic and genotypic analysis of infants with severe respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants. Reduced numbers of NK cells have been reported in infants with severe RSV infection; however, the precise role of NK cells during acute RSV infection is unclear. In this study the NK and T cell phenotypes, LILRB1 gene polymorphisms and KIR genotypes of infants hospitalized with RSV infection were analyzed. Compared to controls, infants with acute RSV infection showed a higher proportion of LILRB1+ T cells; in addition, a subgroup of infants with RSV infection showed an increase in LILRB1+ NK cells. No differences in NKG2C, NKG2A, or CD161 expression between RSV infected infants and controls were observed. LILRB1 genotype distribution of the rs3760860 A>G, and rs3760861 A>G single nucleotide polymorphisms differed between infants with RSV infection and healthy donors, whereas no differences in any of the KIR genes were observed. Our results suggest that LILRB1 participates in the pathogenesis of RSV infection. Further studies are needed to define the role of LILRB1+ NK in response to RSV and to confirm an association between LILRB1 polymorphisms and the risk of severe RSV infection.     

The multifunctional autophagy pathway in the human malaria parasite,Plasmodium falciparum

Autophagy is a catabolic pathway typically induced by nutrient starvation to recycle amino acids, but can also function in removing damaged organelles. In addition, this pathway plays a key role in eukaryotic development. To date, not much is known about the role of autophagy in apicomplexan parasites and more specifically in the human malaria parasite Plasmodium falciparum. Comparative genomic analysis has uncovered some, but not all, orthologs of autophagy-related (ATG) genes in the malaria parasite genome. Here, using a genome-wide in silico analysis, we confirmed that ATG genes whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. We subsequently focused on the molecular and cellular function of P. falciparum ATG8 (PfATG8), an autophagosome membrane marker and key component of the autophagy pathway, throughout the parasite asexual and sexual erythrocytic stages. In this context, we showed that PfATG8 has a distinct and atypical role in parasite development. PfATG8 localized in the apicoplast and in vesicles throughout the cytosol during parasite development. Immunofluorescence assays of PfATG8 in apicoplast-minus parasites suggest that PfATG8 is involved in apicoplast biogenesis. Furthermore, treatment of parasite cultures with bafilomycin A₁ and chloroquine, both lysosomotropic agents that inhibit autophagosome and lysosome fusion, resulted in dramatic morphological changes of the apicoplast, and parasite death. Furthermore, deep proteomic analysis of components associated with PfATG8 indicated that it may possibly be involved in ribophagy and piecemeal microautophagy of the nucleus. Collectively, our data revealed the importance and specificity of the autophagy pathway in the malaria parasite and offer potential novel therapeutic strategies.     

A proteolytic cleavage assay to monitor autophagy in Dictyostelium discoideum

Dictyostelium discoideum is a good model of autophagy. However, the lack of autophagic flux techniques hinders the assessment of new mutants or drugs. One of these techniques, which has been used successfully in yeast and mammalian cells, but has not yet been described in Dictyostelium, is based on the presence of proteolytic fragments derived from autophagic degradation of expressed fusion proteins. Lysosomotropic agents such as NH 4Cl penetrate acidic compartments and raise their pH, thus allowing the accumulation and measurement of these cleaved fragments, which otherwise would be rapidly degraded. We have used this property to detect the presence of free GFP fragments derived from the fusion protein GFP-Tkt-1, a cytosolic marker. We demonstrate that this proteolytic event is dependent on autophagy and can be used to detect differences in the level of autophagic flux among different mutant strains. Moreover, treatment with NH4Cl also facilitates the assessment of autophagic flux by confocal microscopy using the marker RFP-GFP-Atg8.     

High resolution analysis of snake venom metalloproteinase (SVMP) peptide bond cleavage specificity using proteome based peptide libraries and mass spectrometry

Both serine and metalloproteinases have been shown to play the role of toxins in the venoms of many snakes. Determination of the natural protein substrates of these toxins is an important feature in the toxinological characterization of these proteinases. Furthermore, characterization of their peptide bond specificity is of value for understanding active site preference of the proteinase associated with effective proteolysis as well as of use in the design of peptide substrates and inhibitor lead compounds. Typically the determination of peptide bond cleavage specificity of snake venom serine proteinases (SVSPs) and snake venom metalloproteinases (SVMPs) has been performed using limited sets of peptides or small oligopeptides as experimental substrates. Although this approach has yielded valuable data it is generally limited in scope due to the relatively small sets of substrates used to generate the consensus specificity sequences for these proteinases. In this study we use a large, plasma based, proteome-derived peptide library as substrates along with mass spectrometry to explore the peptide bond specificity of three PI SVMPs and one PIII SVMP to determine their individual peptide cleavage consensus sequences. All of the proteinases assayed displayed a clear preference for a leucine residue in the P1' site. Careful analysis of the specificity profiles of the SVMPs examined showed interesting differences in the preferences at the other P and P' sites suggesting functional differences between these proteinases. The PI SVMPs, leucurolysin-a, atrolysin C, and BaP1, showed preferences across the full P4 to P4' range whereas the PIII SVMP bothropasin showed a narrower range of preferences across the sites. In silico docking experiments with the experimentally derived consensus sequences as well as with comparison of the results to those in the literature regarding peptide bond specificity based on both peptide and protein substrates give rise to a fresh understanding of the specificity of these SVMPS and may serve as a foundation for future experiments to better elucidate their mechanism of action in the complex pathophysiology of snakebite envenomation.     

Key events in microvascular damage induced by snake venom hemorrhagic metalloproteinases

Hemorrhage is one of the most significant effects in envenomings induced by viperid snakebites. Damage to the microvasculature, induced by snake venom metalloproteinases (SVMPs), is the main event responsible for this effect. The precise mechanism by which SVMPs disrupt the microvasculature has remained elusive, although recent developments provide valuable clues to deciphering the details of this pathological effect. The main targets of hemorrhagic SVMPs are components of basement membrane (BM) and surrounding extracellular matrix (ECM), which provide mechanical stability to capillaries. P-III SVMPs, comprising disintegrin-like and cysteine-rich domains in addition to the catalytic domain, are more potent hemorrhagic toxins than P-I SVMPs, constituted only by the metalloproteinase domain. This is likely due to the presence of exosites in the additional domains, which contribute to the binding of SVMPs to relevant targets in the microvasculature. Recent in vivo studies have shown that P-III SVMPs are preferentially located in microvessels. On the other hand, the structural determinants responsible for the different hemorrhagic potential of P-I SVMPs remain largely unknown, although backbone flexibility in a loop located near the active site is likely to play a role. Moreover, hemorrhagic and non-hemorrhagic SVMPs differ in their capacity to hydrolyze in vivo key BM proteins, such as type IV collagen and perlecan, as well as other ECM proteins, like types VI and XV collagens, which play a critical role by connecting BM components to perivascular fibrillar collagens. The evidence gathered support a two-step model for the pathogenesis of SVMP-induced hemorrhage: initially, hemorrhagic SVMPs bind to and hydrolyze components of the BM and associated extracellular matrix proteins that play a key role in the mechanical stability of BM. In conditions of normal blood flow in the tissues, such cleavage results in the weakening, distension and eventual disruption of capillary wall due to the action of biophysical forces operating in vivo.     

South American Plasmodium falciparum after the malaria eradication era: clonal population expansion and survival of the fittest hybrids

Malaria has reemerged in many regions where once it was nearly eliminated. Yet the source of these parasites, the process of repopulation, their population structure, and dynamics are ill defined. Peru was one of malaria eradication's successes, where Plasmodium falciparum was nearly eliminated for two decades. It reemerged in the 1990s. In the new era of malaria elimination, Peruvian P. falciparum is a model of malaria reinvasion. We investigated its population structure and drug resistance profiles. We hypothesized that only populations adapted to local ecological niches could expand and repopulate and originated as vestigial populations or recent introductions. We investigated the genetic structure (using microsatellites) and drug resistant genotypes of 220 parasites collected from patients immediately after peak epidemic expansion (1999-2000) from seven sites across the country. The majority of parasites could be grouped into five clonal lineages by networks and AMOVA. The distribution of clonal lineages and their drug sensitivity profiles suggested geographic structure. In 2001, artesunate combination therapy was introduced in Peru. We tested 62 parasites collected in 2006-2007 for changes in genetic structure. Clonal lineages had recombined under selection for the fittest parasites. Our findings illustrate that local adaptations in the post-eradication era have contributed to clonal lineage expansion. Within the shifting confluence of drug policy and malaria incidence, populations continue to evolve through genetic outcrossing influenced by antimalarial selection pressure. Understanding the population substructure of P. falciparum has implications for vaccine, drug, and epidemiologic studies, including monitoring malaria during and after the elimination phase.     

Luteal function,largest follicle,and fertility in postpartum dairy cows treated with 14dCIDR-PGF2α versus 2xPGF2α-Ovsynch for timed AI

A method for timed artificial insemination (AI) that is used for beef cows, beef heifers, and dairy heifers employs progesterone-releasing inserts, such as the controlled internal drug release (CIDR; Zoetis, New York, NY, USA) that are left in place for 14 days. The 14-day CIDR treatment is a method of presynchronization that ensures that cattle are in the late luteal phase of the estrous cycle when PGF_2α is administered before timed AI. The objective of this study was to test the effectiveness of the 14dCIDR-PGF_2α program in postpartum dairy cows by comparing it with the traditional “Presynch-Ovsynch” (2xPGF_2α-Ovsynch) program. The 14dCIDR-PGF_2α cows (n = 132) were treated with a CIDR insert on Day 0 for 14 days. At 19 days after CIDR removal (Day 33), the cows were treated with a luteolytic dose of PGF_2α, 56 hours later were treated with an ovulatory dose of GnRH (Day 35), and 16 hours later were inseminated. The 2xPGF_2α-Ovsynch cows were treated with a luteolytic dose of PGF_2α on Day 0 and again on Day 14. At 12 days after the second PGF_2α treatment (Day 26), the cows were treated with GnRH. At 7 days after GnRH, the cows were treated with PGF_2α (Day 33), then 56 hours later treated with GnRH (Day 35), and then 16 hours later were inseminated. There was no effect of treatment or treatment by parity interaction on pregnancies per AI (P/AI) when pregnancy diagnosis was performed on Day 32 (115/263; 43.7%) or Days 60 to 90 (99/263; 37.6%) after insemination. There was an effect of parity (P < 0.05) on P/AI because primiparous cows had lesser P/AI (35/98; 35.7%) than multiparous cows (80/165; 48.5%) on Day 32. Cows observed in estrus after the presynchronization step (within 5 days after CIDR removal or within 5 days after the second PGF_2α treatment) had greater P/AI than those not observed in estrus (55/103; 53.4% vs. 60/160; 37.5%; observed vs. not observed; P < 0.01; d 32 pregnancy diagnosis). When progesterone data were examined in a subset of cows (n = 208), 55.3% of cows had a “prototypical” response to treatment (i.e., the cow had an estrous cycle that was synchronized by the presynchronization treatment and then the cow responded appropriately to the subsequent PGF_2α and GnRH treatments before timed AI). Collectively, cows with a prototypical response to either treatment had 52.2% P/AI that was greater (P < 0.001) than the P/AI for cows that had a nonprototypical response (19%) (P/AI determined at 60–90 days of pregnancy). In conclusion, we did not detect a difference in P/AI when postpartum dairy cows were treated with 14dCIDR-PGF_2α or 2xPGF2α-Ovsynch before timed AI. The primary limitation to the success of either program was the failure of the cow to respond appropriately to the sequence of treatments.     

Population genetic structure of the Plasmodium vivax circumsporozoite protein (Pvcsp) in Sri Lanka

Molecular methods elucidate evolutionary and ecological processes in parasites, where interaction between hosts and parasites enlighten the evolution of parasite lifestyles and host defenses. Population genetics of Plasmodium vivax parasites accurately describe transmission dynamics of the parasites and evaluation of malaria control measures. As a first generation vaccine candidate against malaria, the Circumsporozoite Protein (CSP) has demonstrated significant potential in P. falciparum. Extensive polymorphism hinders the development of a potent malaria vaccine. Hence, the genetic diversity of Pvcsp was investigated for the first time in 60 Sri Lankan clinical isolates by obtaining the nucleotide sequence of the central repeat (CR) domain and examining the polymorphism of the peptide repeat motifs (PRMs), the genetic diversity indices and phylogenetic relationships. PCR amplicons determined size polymorphism of 610, 700 and 710 bp in Pvcsp of Sri Lanka where all amino acid sequences obtained were of the VK210 variant, consisting variable repeats of 4 different PRMs. The two most abundant PRMs of the CR domain, GDRADGQPA and GDRAAGQPA consisted ~ 2-4 repeats, while GNRAAGQPA was unique to the island. Though, different nucleotide sequences termed repeat allotypes (RATs) were observed for each PRM, these were synonymous contributing to a less polymorphic CR domain. The genetic diversity of Pvcsp in Sri Lanka was due to the number of repetitive peptide repeat motifs, point mutations, and intragenic recombination. The 19 amino acid haplotypes defined were exclusive to Sri Lanka, whereas the 194 Pvcsp sequences of global isolates generated 57 more distinct a.a. haplotypes of the VK210 variant. Strikingly, the CR domain of both VK210 and VK247 variants was under purifying selection interpreting the scarcity of CSP non-synonymous polymorphisms. Insights to the distribution of RATs in the CR region with geographic clustering of the P. vivax VK210 variant were revealed. The cladogram reiterated this unique geographic clustering of local (VK210) and global isolates (VK210 and VK247), which was further validated by the elevated fixation index values of the VK210 variant.     

Detection of Soluble Antigen and DNA of Trypanosoma cruzi in Urine Is Independent of Renal Injury in the Guinea Pig Model

The diagnosis of Chagas disease in humans is generally limited to the detection of specific antibodies. Detection of T. cruzi antigens in urine has been reported previously, but is not used in the diagnosis. In this study, soluble T. cruzi antigens and DNA were detected in urine samples and were associated with kidney injury and systemic detection of the parasite. We used 72 guinea pigs infected with T. cruzi Y strain and 18 non-infected guinea pigs. Blood, kidney, heart and urine samples were collected during the acute phase and chronic phase. Urine samples were concentrated by ultrafiltration. Antigens were detected by Western Blot using a polyclonal antibody against trypomastigote excretory-secretory antigen (TESA). T. cruzi DNA was detected by PCR using primers 121/122 and TcZ1/TcZ2. Levels of T. cruzi DNA in blood, heart and kidney were determined by quantitative PCR. T. cruzi antigens (75 kDa, 80 kDa, 120 kDa, 150 kDa) were detected in the acute phase (67.5%) and the chronic phase (45%). Parasite DNA in urine was detected only in the acute phase (45%). Kidney injury was characterized by high levels of proteinuria, kidney injury molecule-1 (KIM-1) and urea, and some histopathological changes such as inflammation, necrosis, fibrosis and scarce parasites. The detection of antigens and DNA in urine was associated with the presence of parasite DNA in blood and heart and with high levels of parasite DNA in blood, but not with the presence of parasite in kidney or kidney injury. These results suggest that the detection of T. cruzi in urine could be improved to be a valuable method for the diagnosis of Chagas disease, particularly in congenital Chagas disease and in immunocompromised patients.