Humoral immune responses against the malaria vaccine candidate antigen Plasmodium vivax AMA-1 and IL-4 gene polymorphisms in individuals living in an endemic area of the Brazilian Amazon

BackgroundSeveral studies have recently demonstrated that the immune responses against malaria is governed by different factors, including the genetic components of the host. The IL-4 gene appears to be a strong candidate factor because of its role in the regulation of the Th2 response. The present study investigated the role of IL-4 polymorphisms in the development of IgG antibodies against PvAMA-1 and the IL-4 levels in individuals infected with Plasmodium vivax in a malaria endemic area in the Brazilian Amazon.MethodsThe study sample included 83 patients who were diagnosed with P. vivax infection using thick smear and confirmed by nested-PCR. The IL-4 −590 C>T and IL-4 −33 C>T polymorphisms were genotyped by PCR–RFLP, and the intron 3 VNTR was genotyped by PCR. A standardised ELISA protocol was used to measure the total IgG against PvAMA-1. The cytokine/chemokine levels were measured using a Milliplex multiplex assay (Millipore). All of the subjects were genotyped with 48 ancestry informative markers to determine the proportions of African, European and Amerindian ancestry using STRUCTURE software.ResultsOf the 83 patients, 60 (73%) produced IgG antibodies against PvAMA-1. A significant decrease in the percentage of respondents was observed among the primo-infected individuals. No significant differences were observed in the frequencies of genotypes and haplotypes among individuals who were positive or negative for IgG antibodies against PvAMA-1. Furthermore, no significant correlation was observed between the IL-4 polymorphisms, antibody levels, IL-4 levels, and parasitemia.ConclusionsThis study indicated that the polymorphisms identified in the IL-4 gene are not likely to play a role in the regulation of the antibody response against PvAMA-1 and IL-4 production in vivax malaria.     

Investigation on Plasmodium falciparum and Plasmodium vivax infection influencing host haematological factors in tribal dominant and malaria endemic population of Jharkhand

The study was undertaken to elucidate the association of host haematological and biochemical indices in Plasmodium falciparum and Plasmodium vivax malaria in order to explore whether these parameters are unique to disease or act as a potential diagnostic marker.Haematological and biochemical parameters in 106 malarial patients and 33 healthy subjects were evaluated.Following parameters were significantly lower in all infection types (P. vivax, P. falciparum and mixed infection); haemoglobin, blood sugar, PCV and blood urea, while ESR is significantly higher in all types of infection whereas serum bilirubin and creatinine are significantly higher except mixed and vivax infection, respectively. Interestingly, parasitaemia, temperature and age are significantly correlated with blood urea, blood sugar and ESR respectively in vivax infection whereas parasitaemia with PCV and blood sugar and age with PCV in falciparum infection.Malaria infected subjects exhibited alterations in some haematological parameters with low haemoglobin, blood sugar and PCV whereas elevated ESR and serum bilirubin being the important findings observed in our study. These evaluations could be considered to be reliable clinical and biochemical markers for promising diagnostic potential during clinical malarial infection in combination with other genetic and classical microscopic parameters. Haematological evaluation could help in prompt and accurate diagnosis and prevent disease progression by facilitating physicians in clinical correlation for better drug regime.     

Haplotypes of IL12B promoter polymorphisms condition susceptibility to severe malaria and functional changes in cytokine levels in Thai adults

Polymorphic variability in immune response genes, such as IL12B, encoding the IL-12p40 subunit is associated with susceptibility to severe malaria in African populations. Since the role of genetic variation in conditioning severe malaria in Thai adults is largely unexplored, the functional association between IL12B polymorphisms [i.e. IL12Bpro (rs17860508) and IL12B 3′ UTR T/G (rs3212227)], severe malaria and cytokine production was examined in patients with Plasmodium falciparum infections (n = 355) recruited from malaria endemic areas along the Thai–Myanmar border in northwest Thailand. Circulating IL-12p40 (p = 0.049) and IFN-γ (p = 0.051) were elevated in patients with severe malaria, while only IL-12p40 was significantly higher in severe malaria patients with hyperparasitaemia (p = 0.046). Carriage of the IL12Bpro1.1 genotype was associated with enhanced severity of malaria (OR, 2.34; 95% CI, 0.94–5.81; p = 0.066) and hyperparasitaemia (OR, 3.42; 95% CI, 1.17–9.87; p = 0.025) relative to the IL12Bpro2.2 genotype (wild type). Individuals with the IL12Bpro1.1 genotype also had the lowest IL-12p40 (p = 0.002) and the highest IFN-γ (p = 0.004) levels. Construction of haplotypes revealed that carriage of the IL12Bpro-2/3′ UTR-T haplotype was associated with protection against severe malaria (OR, 0.51; 95% CI, 0.29–0.90; p = 0.020) and reduced circulating IFN-γ (p = 0.06). Thus, genotypic and haplotypic variation at IL12Bpro and IL12B 3′ UTR in this population influences susceptibility to severe malaria and functional changes in circulating IL-12p40 and IFN-γ levels. Results presented here suggest that protection against severe malaria in Thai adults is associated with genotypic variants that condition enhanced IL-12p40 and reduced IFN-γ levels.     

IL1B gene polymorphisms influence the course and severity of inflammatory bowel disease

 There is evidence of a disbalance in the inflammatory regulation of patients with inflammatory bowel diseases (IBD). Interleukin-1β plays an important role in the pro-inflammatory response. Our aim was to study the influence which IL1B gene polymorphisms may have on the severity and course of these diseases. Ninety-six patients with ulcerative colitis (UC), 98 patients with Crohn's disease (CD), and 132 ethnically matched healthy individuals (HC) were typed for the polymorphic sites in the promoter region (position –511) and in exon 5 (position +3953) of the IL1B gene, using polymerase chain reaction (PCR)-based methods. In the CD group a significant association (P=0.009) was found in this pair of genes. Homozygotes for allele 1 at position +3953 were more often present (69% vs 31%) in the subgroup of patients carrying at least one copy of allele 2 at position –511. This association was significant in patients with non-perforating disease (P=0.002), but was not present in patients with perforating-fistulizing disease. The distribution of both allelic pairs in the non-fistulizing group proved to be significantly different from HC (P<0.05), UC (P<0.03), and the fistulizing group (P<0.05). There was a similar association in non-operated patients (P=0.024), whereas no such association was found in surgically treated patients. Among carriers of allele 2 at position –511, UC patients with more severe bleeding symptoms (P=0.006) were less frequently found. These results suggest that IL1B gene polymorphisms participate in determining the course and severity of inflammatory bowel disease and contribute to explain the heterogeneity of these diseases. Received: 16 July 1998 / Revised: 3 November 1998     

Association of IL12B polymorphisms with susceptibility to Graves ophthalmopathy in a Taiwan Chinese population

Background

Interleukin 12B (IL12B) gene polymorphisms have been linked to several inflammatory diseases, but their role in the development of Graves ophthalmopathy (GO) in Graves disease (GD) patients is unclear. The purpose of this study was to investigate the disease association of IL12B single nucleotide polymorphisms (SNPs).

Methods

A Taiwan Chinese population comprising 200 GD patients with GO and 271 GD patients without GO was genotyped using an allele-specific extension and ligation method. Hardy-Weinberg equilibrium was estimated using the chi-square test. Allele and genotype frequencies were compared between GD patients with and without GO using the chi-square test.

Results

The genotype and allele frequencies of examined SNPs did not differ between GD patients with and without GO. Although the genotype distribution remained nonsignificant in the sex-stratified analyses, the frequency of the T allele at SNP rs1003199 was significantly higher in patients with GO in the male cohort (P = 6.00 × 10^-3). In addition, haplotypes of IL12B may be used to predict the risk of GO (P = 1.70 × 10^-2); however, we could not prove the statistical significance of analysis after applying the Bonferroni correction.

Conclusions

Our results provide new information that the examined IL12B gene polymorphisms may be associated with susceptibility to GO in the Taiwan Chinese population in a sex-specific manner. This conclusion requires further investigation.     

Cloning, sequence and expression of the lactate dehydrogenase gene from the human malaria parasite, Plasmodium vivax

Increased drug resistance to anti-malarials highlights the need for the development of new therapeutics for the treatment of malaria. To this end, the lactate dehydrogenase (LDH) gene was cloned and sequenced from genomic DNA of Plasmodium vivax ( PvLDH) Belem strain. The 316 amino acid protein-coding region of the PvLDH gene was inserted into the prokaryotic expression vector pKK223-3 and a 34 kDa protein with LDH activity was expressed in E. coli. Structural differences between human LDHs and PfLDH make the latter an attractive target for inhibitors leading to novel anti-malarial drugs. The sequence similarity between PvLDH and PfLDH (90% residue identity and no insertions or deletions) indicate that the same approach could be applied to Plasmodium vivax, the most common human malaria parasite in the world.     

Retinol supplementation in murine Plasmodium berghei malaria: effects on tissue levels, parasitaemia and lipid peroxidation

Reduced plasma retinol concentrations occur in human malaria but the benefits of supplementation remain uncertain. We assessed the in vivo efficacy of retinol administration, and its effect on lipid peroxidation, in a Plasmodium berghei murine model. Animals received vehicle (n=17) or retinol (i) before P. berghei inoculation (four doses), (ii) at parasitaemia 10-15% (three to four doses) or (iii) before and after inoculation (six to seven doses; n=15 in each group), with euthanasia on day 8 post-inoculation or when the parasitaemia exceeded 50%. Multiple-dose pre-inoculation retinol reduced endpoint parasitaemia by 24% (P=0.001 versus controls). A reduction of 18% (P=0.042) was observed when retinol was given to parasitaemic animals. Retinol was ineffective when given both before and after infection (11% reduction; P=0.47). Although retinol supplementation did not change plasma retinol concentrations, liver retinol content increased and correlated inversely with endpoint parasitaemia (r=-0.45, P=0.001). Malaria infection augmented concentrations of the free radical lipid peroxidation end-product F(2)-isoprostanes in plasma, erythrocytes and liver by 1.8-, 2.8- and 4.9-fold, respectively, but retinol supplementation had no effect on these increases. Consistent with some human malaria studies, prophylactic retinol reduces P. berghei parasitaemia. This effect relates to augmentation of tissue retinol stores rather than to retinol-associated changes in oxidant status.     

Variation in relapse frequency and the transmission potential of Plasmodium vivax malaria

There is substantial variation in the relapse frequency of Plasmodium vivax malaria, with fast-relapsing strains in tropical areas, and slow-relapsing strains in temperate areas with seasonal transmission. We hypothesize that much of the phenotypic diversity in P. vivax relapses arises from selection of relapse frequency to optimize transmission potential in a given environment, in a process similar to the virulence trade-off hypothesis. We develop mathematical models of P. vivax transmission and calculate the basic reproduction number R₀ to investigate how transmission potential varies with relapse frequency and seasonality. In tropical zones with year-round transmission, transmission potential is optimized at intermediate relapse frequencies of two to three months: slower-relapsing strains increase the opportunity for onward transmission to mosquitoes, but also increase the risk of being outcompeted by faster-relapsing strains. Seasonality is an important driver of relapse frequency for temperate strains, with the time to first relapse predicted to be six to nine months, coinciding with the duration between seasonal transmission peaks. We predict that there is a threshold degree of seasonality, below which fast-relapsing tropical strains are selected for, and above which slow-relapsing temperate strains dominate, providing an explanation for the observed global distribution of relapse phenotypes.     

Plasmodium berghei: development of an irreversible experimental malaria model in Wistar rats

A protocol to infect five-week-old Wistar rats by Plasmodium berghei which resulted in 100% mortality was developed in this work. In order to accomplish this goal, the effect of the administration of 10(7) and 10(8) parasitized erythrocytes by i.v. and i.p. route was investigated. The animals inoculated with 10(7) parasitized red blood cells by i.p. and i.v. routes showed 25 and 50% mortality, respectively. Inoculation with 10(8) parasitized erythrocytes by both routes resulted in a 100% lethal infection. The i.v. inoculation showed less scattered results and it was preferred over the i.p. route. The suitability of the protocol developed was evaluated by treating infected Wistar rats with chloroquine (30 mg/kg/day). A decreased parasitemia after the treatment was observed until the complete eradication of the parasite, around 10 days post-inoculation. Parasitemia depression after chloroquine treatment demonstrates the utility of the model developed to test new antimalarial drugs.     

IL1β, IL6 and IL8 gene polymorphisms involvement in recurrent corneal erosion in patients with hereditary stromal corneal dystrophies

TGFBI gene mutations cause corneal stromal dystrophies of autosomal dominant inheritance. The most frequent complication of stromal dystrophies is recurrent corneal erosion with varying degree of accompanying inflammation. IL-1, IL-6 and IL-8 are main cytokines involved in corneal erosion healing. This study aimed to investigate the association between IL1B gene ?511C/T, IL6 gene ?174G/C and IL8 gene ?781C/T polymorphisms and risk of recurrent erosion development in patients with hereditary corneal stromal dystrophies. A trend to decrease of IL1B gene ?511TT genotype frequency in group with erosion (3.7%) comparing to control (6.7%) was observed. IL6 gene ?174C allele carriers frequency in control group (65.9%) was significantly (P < 0.05) lower comparing to patients with erosion (80.5%). Frequency of IL8 ?781TT genotype was significantly (P < 0.05) lower in the group with erosion (10.7%) comparing to patients without erosion (30.8%) and control (25%). IL6 gene ?174C allele may be considered as genetic marker of corneal erosion risk in patients with hereditary stromal corneal dystrophies, whereas IL8 ?781TT genotype is associated with negative recurrent erosion prognosis in such patients.     

Features and prognosis of severe malaria caused by Plasmodium falciparum, Plasmodium vivax and mixed Plasmodium species in Papua New Guinean children

Background

Mortality from severe pediatric falciparum malaria appears low in Oceania but Plasmodium vivax is increasingly recognized as a cause of complications and death. The features and prognosis of mixed Plasmodium species infections are poorly characterized. Detailed prospective studies that include accurate malaria diagnosis and detection of co-morbidities are lacking.

Methods and Findings

We followed 340 Papua New Guinean (PNG) children with PCR-confirmed severe malaria (77.1% P. falciparum, 7.9% P. vivax, 14.7% P. falciparum/vivax) hospitalized over a 3-year period. Bacterial cultures were performed to identify co-incident sepsis. Clinical management was under national guidelines. Of 262 children with severe falciparum malaria, 30.9%, 24.8% and 23.2% had impaired consciousness, severe anemia, and metabolic acidosis/hyperlactatemia, respectively. Two (0.8%) presented with hypoglycemia, seven (2.7%) were discharged with neurologic impairment, and one child died (0.4%). The 27 severe vivax malaria cases presented with similar phenotypic features to the falciparum malaria cases but respiratory distress was five times more common (P = 0.001); one child died (3.7%). The 50 children with P. falciparum/vivax infections shared phenotypic features of mono-species infections, but were more likely to present in deep coma and had the highest mortality (8.0%; P = 0.003 vs falciparum malaria). Overall, bacterial cultures were positive in only two non-fatal cases. 83.6% of the children had alpha-thalassemia trait and seven with coma/impaired consciousness had South Asian ovalocytosis (SAO).

Conclusions

The low mortality from severe falciparum malaria in PNG children may reflect protective genetic factors other than alpha-thalassemia trait/SAO, good nutrition, and/or infrequent co-incident sepsis. Severe vivax malaria had similar features but severe P. falciparum/vivax infections were associated with the most severe phenotype and worst prognosis.     

Microsatellite variation, repeat array length, and population history of Plasmodium vivax

A recent paper (Leclerc et al. 2004) described limited variationin dinucleotide microsatellites from Plasmodium vivax, suggestingvery recent bottlenecks or genome-wide selective events. Wedescribe patterns of variation in 11 dinucleotide microsatellitesin P. vivax populations from Colombia, India, and Thailand.We find abundant variation with heterozygosity of 0.64, 0.76,and 0.77, respectively, in the three countries. The discrepancybetween these two studies results is simply explained by thedifferences in the size of repeat arrays. The microsatellitesstudied by Leclerc et al. (2004) have very few repeats (median5.5, range 4–13) and so would not be expected to be variable.Plasmodium vivax microsatellites show comparable levels of variationto those in Plasmodium falciparum when repeat array length istaken into account and provide no support for recent bottlenecksor widespread selective purging     

Cloning, expression, and characterisation of a Plasmodium vivax MSP7 family merozoite surface protein

Plasmodium vivax remains the most widespread Plasmodium parasite species around the world, producing about 75 million malaria cases, mainly in South America and Asia. A vaccine against this disease is of urgent need, making the identification of new antigens involved in target cell invasion, and thus potential vaccine candidates, a priority. A protein belonging to the P. vivax merozoite surface protein 7 (PvMSP7) family was identified in this study. This protein (named PvMSP7(1)) has 311 amino acids displaying an N-terminal region sharing high identity with P. falciparum MSP7, as well as a similar proteolytical cleavage pattern. This protein's expression in P. vivax asexual blood stages was revealed by immuno-histochemical and molecular techniques.     

Association of polymorphisms of cytokine genes (IL1B, IL1RN, TNFA, LTA, IL6, IL8, and IL10) with chronic obstructive pulmonary disease

To assess the role that polymorphisms of cytokine genes play in genetic predisposition to chronic obstructive pulmonary disease (COPD), the allele and genotype distributions of IL1B, IL1RN, TNFA, LTA, IL6, IL8, and IL10 were studied in COPD patients (N = 319) and healthy individuals (N = 403), residents of Ufa, Bashkortostan. Genotype IL1RN*2/IL1RN*2 of IL1RN was identified as a risk factor for COPD, its frequency being 9.80% in the COPD patients and 4.67% in the healthy subjects (x ² = 5.45, df = 1, P = 0.02, OR = 2.21). Genotype GG of the LTA polymorphism A252G was significantly more common in the COPD patients than in the controls (7.84% vs. 3.72%; x ² = 5.00, df = 1, P = 0.026). In patients with COPD stage IV, the frequency of this genotype was twice as high as in those with COPD stages II and III (11.18% vs. 4.79%; x ² = 3.08, df = 1, P = 0.08). Genotype GG of the TNFA polymorphism G(?308)A in combination with genotype AA of the LTA polymorphism A252G was significantly less frequent in the COPD patients than in the healthy subjects (38.55% vs. 46.93%; x ² = 8.82, df = 1, P = 0.0039). Genotype GG of the IL6 polymorphism G(?174)C was more frequent in the patients with COPD stage IV (43.75% vs. 31.54% in the patients with COPD stages II and III, x ² = 4.15, P = 0.042). No significant differences were found between the groups of COPD patients and healthy subjects concerning the genotype frequencies of the polymorphisms T(?511)C and T3953C of IL1B, G(?308)A of TNFA, G(?174)C of IL6, A(?251)C of IL8, and C(?627)A of IL10.     

PPARA, RXRA, NR1I2 and NR1I3 gene polymorphisms and lipid and lipoprotein levels in a Southern Brazilian population

Cardiovascular disease is the main cause of death worldwide, and dyslipidemia is an important multifactorial risk factor. Considering the involvement of nuclear receptors in metabolic pathways, and that some of the receptors act in lipid metabolism and homeostasis, the aim of the present study was to investigate the influence of genetic variations in RXRA, PPARA, NR1I2, and NR1I3 on lipid and lipoprotein levels. Five polymorphisms in the aforementioned genes were genotyped in 622 Brazilians of European descent by PCR-RFLP or TaqMan genotyping assays. In general, carriers of the A insertion of RXRA rs11381416 polymorphism showed higher levels of triglyceride (TG; 1.80 ± 1.20 vs. 1.52 ± 1.20 mmol/L; P = 0.020). Moreover, sexual dimorphic association was found (gender*NR1I3 rs2501873 genotype interaction P < 0.001), males with NR1I3 rs2501873 G/G genotype had lower TG levels (ANCOVA, P = 0.009). Our results suggest that polymorphisms in the RXRA and NR1I3 genes influence lipid profile in a Southern Brazilian population. However, these general and gender association require confirmation in subsequent studies.     

IL28B gene polymorphisms and Th1/Th2 cytokine levels might be associated with HTLV-associated arthropathy

The present study is the first investigation of the association between single nucleotide polymorphisms (SNPs – rs8099917, rs12979860 and rs8103142) of the IL28B gene and the development of human T-lymphotropic virus (HTLV)-associated arthropathy (HAA). Individuals with HAA exhibited low interleukin (IL) 6 (p < 0.05) and high IL-10 (p < 0.05) levels compared with asymptomatic patients. TNF-α/CD4⁺ T cell count, TNF-α/CD8⁺ T cell count and IFN-γ/proviral load positively correlated in asymptomatic patients. The allelic and genotypic frequencies did not differ between patients with HAA and asymptomatic patients. Seven haplotypes were detected in the investigated population, with haplotype CCT (p < 0.05) being the most frequent among the HTLV-infected individuals, while haplotype TTG (p < 0.05) was detected in the group with HAA only. Compared with asymptomatic patients, individuals with HAA and genotype TT (rs8099917) exhibited larger numbers of CD8⁺ T cells (p < 0.05) and higher proviral load levels (p < 0.05). Those patients with HAA and genotypes CC (rs12979860) and TT (rs8103142) exhibited high TNF-β (p < 0.05) and IFN-γ (p < 0.05) levels. Those patients with HAA and genotype CT/TT (rs12979860) exhibited high IL-10 levels (p < 0.05). These results suggest that haplotypes CCT and TTG might be associated with susceptibility to HTLV infection and progression to HAA, respectively. Genotype TT (rs8099917) might be a risk factor for elevation of the proviral load and CD8⁺ T cell count. In addition, genotypes CC (rs12979860) and TT (rs8103142) seem to be associated with increased TNF-β and IFN-γ levels.     

Plasmodium falciparum: effect of radiation on levels of gene transcripts in sporozoites

Humans immunized by the bites of irradiated Plasmodium falciparum (Pf) sporozoite-infected mosquitoes are protected against malaria. Radiation attenuates the sporozoites preventing them from fully developing and replicating in hepatocytes, but the effects of radiation on gene expression in sporozoites are unknown. We used RT-PCR (35 cycles of PCR followed by densitometry) to assess the expression of ten genes in Pf sporozoites, and in sporozoites irradiated with 15,000cGy. Irradiation reduced expression substantially (>60%) of two DNA repair genes; moderately (30-60%) of PfUIS3, the Pf orthologue of PbUIS3, a gene up-regulated in Plasmodium berghei sporozoites and of a third DNA repair gene; and minimally (<30%) of the Pf18S ribosomal RNA, PfCSP, PfSSP2/TRAP, and PfCELTOS genes. Irradiation increased expression of PfSPATR minimally. PfLSA1 RNA was not detectable in sporozoites. These results establish that radiation of sporozoites affects gene expression levels and provide the foundation for studies to identify specific genes involved in attenuation and protective immunity.