Multiplex sequence-specific polymerase chain reaction reveals new MASP2 haplotypes associated with MASP-2 and MAp19 serum levels.

Deficiency of mannan-binding lectin-associated serine protease 2 (MASP-2) has been associated with infections, whereas high levels appear to increase the risk of inflammatory disorders. Nevertheless, MASP2 haplotypes have been poorly investigated. To overcome haplotyping cost and time consumption, we developed multiplex polymerase chain reactions with sequence-specific primers (PCR-SSP) for 8 single nucleotide polymorphisms (SNPs), reducing the number of necessary reactions from 18 to 7. SNPs were distributed from the promoter to the last exon, and a single PCR-SSP was used for p.D120G. We evaluated the phylogenetic relationships and global distribution of 10 identified haplotypes in 338 Danish individuals with known MASP-2 and MAp19 levels and 309 South Brazilians. Four haplotypes were associated with reduced MASP-2 levels in plasma (lower than 200 ng/mL). Simultaneous association with the highest MASP-2 (over 600 ng/mL) and lowest MAp19 levels (lower than 200 ng/mL) was demonstrated with the intron 9 mutation (Kruskal-Wallis p < 0.0001). Cumulative genotype frequencies predict approximately 0.4% severely deficient and 25% overproducing individuals in both populations. Rapid and low-cost screening of patients with multiplex MASP2 PCR-SSP could be used to identify clinical conditions where MASP-2 (or MAp19) levels may be disease modifying, possibly improving disease outcome through early therapeutic and preventive measures.

Novel regulatory SNPs in the promoter region of the TNFRSF18 gene in a Gabonese population

Parasites are accountable for driving diversity within immune gene families. We identified and investigated regulatory single nucleotide polymorphisms (SNPs) in the promoter regions of the tumor necrosis factor receptor superfamily member 18 (TNFRSF18) gene by direct sequencing in a group of male Gabonese individuals exposed to a wide array of parasitic diseases such as malaria, filariasis and schistosomiasis. Two new promoter variants were identified in 40 individuals. Both novel variants were heterozygous and were linked to SNP #rs3753344 (C/T), which has been described. One of the SNP variants (ss2080581728) was close to the general transcription factor site, the TATA box. We further validated these new promoter variants for their allelic gene expression using transient transfection assays. One new promoter variant with two base changes (C/T - ss2080581728/rs3753344) displayed an altered expression of the marker gene. Both novel variants remained less active at the non-induced state in comparison to the major allele. The allele frequencies observed in this study were consistent with data for other African populations. The detection and analysis of these human immune gene polymorphisms contribute to a better understanding of the interaction between host-parasite and expression of Treg activity.

Novel regulatory SNPs in the promoter region of the TNFRSF18 gene in a Gabonese population.

Parasites are accountable for driving diversity within immune gene families. We identified and investigated regulatory single nucleotide polymorphisms (SNPs) in the promoter regions of the tumor necrosis factor receptor superfamily member 18 (TNFRSF18) gene by direct sequencing in a group of male Gabonese individuals exposed to a wide array of parasitic diseases such as malaria, filariasis and schistosomiasis. Two new promoter variants were identified in 40 individuals. Both novel variants were heterozygous and were linked to SNP #rs3753344 (C/T), which has been described. One of the SNP variants (ss2080581728) was close to the general transcription factor site, the TATA box. We further validated these new promoter variants for their allelic gene expression using transient transfection assays. One new promoter variant with two base changes (C/T - ss2080581728/rs3753344) displayed an altered expression of the marker gene. Both novel variants remained less active at the non-induced state in comparison to the major allele. The allele frequencies observed in this study were consistent with data for other African populations. The detection and analysis of these human immune gene polymorphisms contribute to a better understanding of the interaction between host-parasite and expression of Treg activity.

Prevalence and association of human parvovirus B19V with hepatitis B and C viruses in Nigeria.

Co-infection of parvovirus B19 with hepatitis B virus has been found in patients with acute and chronic hepatitis. The clinical significance of parvovirus B19 in hepatitis B co-infected patients is still controversial. In this study parvovirus B19 antibodies and DNA were investigated in serum samples from 76 patients with HBV infection, 17 with HBV/HCV co-infection and 44 healthy controls. In the sera from patients with HBV infection, anti-B19V IgM and IgG antibodies were detected in 24/76 (32%) and 25/76 (33%), in 6/17 (35%) and 8/17 (47%) of HBV/HCV co-infected patients, and in 14/44 (32%) and 12/44 (12%) of a non-hepatitis healthy controls, respectively. B19V DNA was detected in 8/76 (11%) of patients with HBV infection and in 3/17 (18%) of patients with a HBV/HCV co-infection, and in 4/44 (9%) healthy controls. The occurrence of parvovirus B19 DNA was significantly higher in patients with symptomatic HBV 4/20 (20%) compared to asymptomatic HBV carrier 4/56 (7%) (P<0.05). Ten of the positive B19V DNA sequences belonged to B19V genotype 1 while two belonged to genotype 3. The results of this study showed a significant difference in the prevalence of parvovirus B19 DNA in symptomatic HBsAg positive as compared to asymptomatic HBsAg positive individuals; however, the conclusion that parvovirus B19 infection increased the frequency of liver disease was not supported. Long-term longitudinal studies are, however, required to determine the synergistic effect of parvovirus B19 infection in HBV or HBV and HCV co-infected persons.

Permanent Genetic Resources added to the Molecular Ecology Resources Database 1 February 2010-31 March 2010.

This article documents the addition of 228 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anser cygnoides, Apodemus flavicollis, Athene noctua, Cercis canadensis, Glis glis, Gubernatrix cristata, Haliotis tuberculata, Helianthus maximiliani, Laricobius nigrinus, Laricobius rubidus, Neoheligmonella granjoni, Nephrops norvegicus, Oenanthe javanica, Paramuricea clavata, Pyrrhura orcesi and Samanea saman. These loci were cross-tested on the following species: Apodemus sylvaticus, Laricobius laticollis and Laricobius osakensis (a proposed new species currently being described).

Ficolin 2 (FCN2) functional polymorphisms and the risk of rheumatic fever and rheumatic heart disease.

Ficolins are pattern-recognition proteins involved in innate immunity, which upon binding to their specific pathogen-associated molecular patterns on the microbial surfaces trigger the immune response either by binding to collectin cellular receptors or by initiating the complement lectin pathway. In humans, three ficolin genes have been identified, which encode ficolin-1 (M-ficolin), ficolin-2 (L-ficolin) and ficolin-3 (H-ficolin or Hakata antigen). Ficolin-2 was shown to bind to lipoteichoic acid, a cell wall constituent in all Gram-positive bacteria such as Streptococcus pyogenes, which is the aetiological agent of rheumatic fever (RF) and its most severe sequelae, chronic rheumatic heart disease (CRHD). Here we investigated polymorphisms in the promoter region of the FCN2 gene (at positions -986/-602 and +4) in 122 patients with RF and CRHD and in 210 healthy subjects from the same geographic region and socioeconomic background. The haplotype -986/-602/-4 G/G/A, which is related to low levels of L-ficolin, was observed more frequently in the CRHD group when compared to the healthy subjects [99/162, 61.1% versus 211/420, 50.2%, odds ratio (OR) 1.6, confidence interval (CI) 95% 1.1-2.3, P = 0.021]. The haplotype -986/-602/-4 A/G/A was observed more frequently in the healthy group when compared to the affected (RF plus CRHD) subjects (31/420, 7.4% versus 6/244, 2.5%, OR 3.2, CI 95% 0.13-0.77, P = 0.008). Based on those findings, one can conclude that polymorphisms associated with low levels of L-ficolin level may predispose an individual to recurrent and/or more severe streptococcal infection.

In vivo anti-malarial effect of the beta-amino alcohol 1t on Plasmodium berghei.

Glycerol derivatives are a class of compounds, which are easy and inexpensive to produce with potent anti-malarial activities against blood stages of Plasmodium falciparum in vitro. In the present study, one of these compounds, termed 1t, which had the lowest IC(50) values, was assessed in a murine malarial model. Nuclear magnetic resonance imaging and Balb/c mice infected with Plasmodium berghei ANKA strain were treated in a 4-day suppressive test. Mice received a once-daily intraperitoneal administration of 50 mg/Kg of the drug for 4 days. Although no parasitaemia clearance was reached, a slower parasite proliferation and a slightly longer survival time compared with the placebo group were observed.

Monoquaternary ammonium derivatives inhibit growth of protozoan parasites.

The phospholipid metabolism of Plasmodium falciparum-infected erythrocytes has been shown to be an effective pharmacological target for novel chemotherapy. Thirty-seven monoquaternary ammonium derivatives analogous to choline were screened for their potential antiprotozoal activity against P. falciparum and Leishmania braziliensis. Twenty-three compounds inhibited chloroquine resistant and sensitive P. falciparum strains with inhibitory concentrations ranging from 0.001 microM to 47 microM. Among the inhibitors were six compounds with nanomolar activity containing at least one ethyl group in the polar head and a hydrophobic alkyl chain with 10 to 14 methylene groups. Four compounds also exhibited in vitro antileishmanial properties in the micromolar range.

Diversity of the MBL2 gene in various Brazilian populations and the case of selection at the mannose-binding lectin locus.

The mannose binding lectin (MBL2) polymorphism is responsible for a common immunodeficiency in the human species. There were suggestions that the MBL2 polymorphism has been under balancing selection, based on the high global frequency of alleles generating MBL deficiency and on the worldwide distribution of diseases negatively associated with them. To describe the distribution of MBL2 allelic haplotypes in Brazilian populations and to discuss the evolution of this polymorphism, we analyzed six South Brazilian populations (152 Guarani Amerindian, 239 Kaingang Amerindian, 107 admixed, Brazilian 32 Afro-Brazilian, 202 Euro-Brazilian and 16 Oriental-Brazilian). Eight haplotypes were observed: MBL2*HYPA, LYQA, LYPA, LXPA, LYPB, LYQC, HYPD, and LYPD. In addition, through sequencing of the promoter and exon 1 from Amerindian and Oriental individuals, three new single-nucleotide polymorphisms (SNPs) were found in the MBL2 promoter region in the Kaingang. Analysis of the sequencing data by neutrality tests (Tajima's D and Fu and Li's D* and F*) revealed no deviation from selective neutrality equilibrium in the Guarani and Kaingang. Significant Fay and Wu's H results are explained by the recent gene flow in these populations. Contrarily to previous thoughts, stochastic evolutionary factors seem therefore to have had a predominant role in shaping the MBL2 polymorphism, at least in the Amerindians.

Association of a new mannose-binding lectin variant with severe malaria in Gabonese children.

Mannose-binding lectin (MBL2) variants that decrease the plasma level of the protein or encode dysfunctional proteins are frequently associated with the severity of a number of infections and autoimmune disorders. The high frequencies of these variants in most populations of the world are probably maintained by some selective advantage against widespread diseases. We found 14 new MBL2 allelic haplotypes, two of them with non-synonymous variants, by screening 136 children with uncomplicated malaria, 131 children with severe malaria and 39 older healthy schoolchildren. We also found a significant association of a novel variant with susceptibility to severe malaria (P=0.010). Increased MBL plasma levels and corresponding MBL2 genotypes were associated with lower concentration of several cytokines and chemokines in plasma of malaria patients. We suggest that malaria could have been one of the evolutionary driving forces shaping the MBL2 polymorphism in the African population.