Association of Toll-like receptor polymorphisms with HIV status in North Americans.

Single-nucleotide polymorphisms (SNPs) in Toll-like receptor (TLR) genes TLR2-4 and TLR7-9, but not in TLR1 and TLR6, have been previously evaluated regarding human immunodeficiency virus (HIV) acquisition and disease progression in various populations, most of which were European. In this study, we examined associations between a total of 41 SNPs in 8 TLR genes (TLR1-4, TLR6-9) and HIV status in North American subjects (total n=276 (Caucasian, n=102; African American, n=150; other, n=24)). Stratification of the data by self-identified race revealed that a total of nine SNPs in TLR1, TLR4, TLR6 and TLR8 in Caucasians, and two other SNPs, one each in TLR4 and TLR8, in African Americans were significantly associated with HIV status at P<0.05. Concordant with the odds ratios of these SNPs, significant differences were observed in the SNP allele frequencies between HIV+ and HIV- subjects. Finally, in Caucasians, certain haplotypes of single (TLR1 and TLR4) and heterodimer (TLR2_TLR6) genes may be inferred as 'susceptible' or 'protective'. Our study provides in-depth insight into the associations between TLR variants, particularly TLR1 and TLR6, and HIV status in North Americans, and suggests that these associations may be race specific.

Variation in human ß-defensin genes: new insights from a multi-population study.

Human ß-defensin 2 (hBD-2) and hBD-3, encoded by DEFB4 and DEFB103A, respectively, have shown anti-HIV activity, and both genes exhibit copy number variation (CNV). Although the role of hBD-1, encoded by DEFB1, in HIV-1 infection is less clear, single nucleotide polymorphisms (SNPs) in DEFB1 may influence viral loads and disease progression. We examined the distribution of DEFB1 SNPs and DEFB4/103A CNV, and the relationship between DEFB1 SNPs and DEFB4/103A CNV using samples from two HIV/AIDS cohorts from the United States (n = 150) and five diverse populations from the Coriell Cell Repositories (n = 46). We determined the frequencies of 10 SNPs in DEFB1 using a post-PCR, oligonucleotide ligation detection reaction-fluorescent microsphere assay, and CNV in DEFB4/103A by real-time quantitative PCR. There were noticeable differences in the frequencies of DEFB1 SNP alleles and haplotypes among various racial/ethnic groups. The DEFB4/103A copy numbers varied from 2 to 8 (median, 4), and there was a significant difference between the copy numbers of self-identified whites and blacks in the US cohorts (Mann-Whitney U-test P = 0.04). A significant difference was observed in the distribution of DEFB4/103A CNV among DEFB1 -52G/A and -390T/A genotypes (Kruskal-Wallis P = 0.017 and 0.026, respectively), while not in the distribution of DEFB4/103A CNV among -52G/A_-44C/G_-20G/A diplotypes. These observations provide additional insights for further investigating the complex interplay between ß-defensin genetic polymorphisms and susceptibility to, or the progression or severity of, HIV infection/disease.

The association of the glycophorin C exon 3 deletion with ovalocytosis and malaria susceptibility in the Wosera, Papua New Guinea.

Erythrocyte polymorphisms, including ovalocytosis, have been associated with protection against malaria. This study in the Wosera, a malaria holoendemic region of Papua New Guinea, examined the genetic basis of ovalocytosis and its influence on susceptibility to malaria infection. Whereas previous studies showed significant associations between Southeast Asian ovalocytosis (caused by a 27- base pair deletion in the anion exchanger 1 protein gene) and protection from cerebral malaria, this mutation was observed in only 1 of 1019 individuals in the Wosera. Polymerase chain reaction strategies were developed to genotype individuals for the glycophorin C exon 3 deletion associated with Melanesian Gerbich negativity (GPCDeltaex3). This polymorphism was commonly observed in the study population (GPCDeltaex3 frequency = 0.465, n = 742). Although GPCDeltaex3 was significantly associated with increased ovalocytosis, it was not associated with differences in either Plasmodium falciparum or P vivax infection measured over the 7-month study period. Future case-control studies will determine if GPCDeltaex3 reduces susceptibility to malaria morbidity.

Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with pfcrt polymorphism in Papua New Guinea and South America.

The mechanistic basis for chloroquine resistance (CQR) in Plasmodium falciparum recently has been linked to the polymorphic gene pfcrt. Alleles associated with CQR in natural parasite isolates harbor threonine (T), as opposed to lysine (K) at amino acid 76. P. falciparum CQR strains of African and Southeast Asian origin carry pfcrt alleles encoding an amino acid haplotype of CVIET (residues 72-76), whereas most South American CQR strains studied carry an allele encoding an SVMNT haplotype; chloroquine-sensitive strains from malarious regions around the world carry a CVMNK haplotype. Upon investigating the origin of pfcrt alleles in Papua New Guinean (PNG) P. falciparum we found either the chloroquine-sensitive-associated CVMNK or CQR-associated SVMNT haplotypes previously seen in Brazilian isolates. Remarkably we did not find the CVIET haplotype observed in CQR strains from Southeast Asian regions more proximal to PNG. Further we found a previously undescribed CQR phenotype to be associated with the SVMNT haplotype from PNG and South America. This CQR phenotype is significantly less responsive to verapamil chemosensitization compared with the effect associated with the CVIET haplotype. Consistent with this, we observed that verapamil treatment of P. falciparum isolates carrying pfcrt SVMNT is associated with an attenuated increase in digestive vacuole pH relative to CVIET pfcrt-carrying isolates. These data suggest a key role for pH-dependent changes in hematin receptor concentration in the P. falciparum CQR mechanism. Our findings also suggest that P. falciparum CQR has arisen through multiple evolutionary pathways associated with pfcrt K76T.

Reciprocal immunomodulatory effects of gamma interferon and interleukin-4 on filaria-induced airway hyperresponsiveness.

Tropical pulmonary eosinophilia (TPE) is a severe asthmatic syndrome of lymphatic filariasis, in which an allergic response is induced to microfilariae (Mf) in the lungs. Previously, in a murine model for TPE, we have demonstrated that recombinant interleukin-12 (IL-12) suppresses pulmonary eosinophilia and airway hyperresponsiveness (AHR) by modulating the T helper (Th) response in the lungs from Th2- to Th1-like, with elevated gamma-interferon (IFN-gamma) production and decreased IL-4 and IL-5 production. The present study examined the immunomodulatory roles of IL-4 and IFN-gamma in filaria-induced AHR and pulmonary inflammation using mice genetically deficient in these cytokines. C57BL/6, IL-4 gene knockout (IL-4(-/-)), and IFN-gamma(-/-) mice were first immunized with soluble Brugia malayi antigens and then inoculated intravenously with 200,000 live Mf. Compared with C57BL/6 mice, IL-4(-/-) mice exhibited significantly reduced AHR, whereas IFN-gamma(-/-) mice had increased AHR. Histopathologically, each mouse strain showed increased cellular infiltration into the lung parenchyma and bronchoalveolar space compared with naïve animals. However, consistent with changes in AHR, IL-4(-/-) mice had less inflammation than C57BL/6 mice, whereas IFN-gamma(-/-) mice had exacerbated pulmonary inflammation with the loss of pulmonary architecture. Systemically, IL-4(-/-) mice produced significantly higher IFN-gamma levels compared with C57BL/6 mice, whereas IFN-gamma(-/-) mice produced significantly higher IL-4 levels. These data indicate that IL-4 is required for the induction of filaria-induced AHR, whereas IFN-gamma suppresses AHR.

Frequent umbilical cord-blood and maternal-blood infections with Plasmodium falciparum, P. malariae, and P. ovale in Kenya.

The prevalence of malaria infection in 102 paired maternal-blood and umbilical cord-blood samples was assessed by microscopy and polymerase chain reaction (PCR) in a holoendemic area in Kenya. Plasmodium falciparum single-species infection was detected in maternal peripheral blood (3.4%), whereas microscopy indicated that no Plasmodium species were in cord blood. In contrast, maternal-blood samples showed a PCR prevalence of 48% for P. falciparum, 25% for P. malariae, and 24% for P. ovale, and cord-blood samples showed a PCR prevalence of 32%, 23%, and 21%, respectively. Although mothers with mixed-species infections were more likely to have offspring infected with mixed species, the specific malaria species were discordant in paired maternal- and cord-blood samples. Triple-species infections were observed in 11 cord- and maternal-blood samples at a 5.5-fold greater frequency than expected. These findings indicate that Plasmodium species infections in cord blood are common, occur at lower densities, and may be acquired before parturition.

Random distribution of mixed species malaria infections in Papua New Guinea.

Plasmodium falciparum (Pf), P. vivax (Pv), P. malariae (Pm), and P. ovale (Po) infections are endemic in coastal areas of Papua New Guinea. Here 2,162 individuals living near Dreikikir, East Sepik Province, have been analyzed for complexity of malaria infection by blood smear and polymerase chain reaction (PCR) diagnoses. According to blood smear, the overall prevalence of Plasmodium infection was 0.320. Most individuals (0.283) were infected with a single species only. The prevalence of mixed species infections was low (0.037). Further analysis of a 173-sample subset by nested PCR of small subunit ribosomal DNA resulted in an overall 3.0-fold increase in prevalence of infection, with a 17.5-fold increase in the frequency of mixed species infections. Among mixed species infections detected by PCR, the frequency of double species was 0.364, and that of triple species was 0.237. Nine individuals (0.052) were infected with all 4 species. To determine if infection status (uninfected, single, and multiple infections) deviates from an independent random distribution (null hypothesis), observed versus expected frequencies of all combinations of Plasmodium species infections, or assemblages (Pf-, Pv-, Pm-, Po-, to Pf+, Pv+, Pm+, Po+), were compared using a multiple-kind lottery model. All 4 species were randomly distributed whether diagnosed by blood smear or PCR in the overall population and when divided into age group categories. These findings suggest that mixed species malaria infections are common, and that Plasmodium species appear to establish infection independent of one another.

Molecular basis of defence against oxidative stress in Entamoeba histolytica and Giardia lamblia.

Gastrointestinal protozoa, viz. Entamoeba histolytica and Giardia, are able to survive in a microaerobic environment. The role of parasitic factors, particularly cysteine, cysteine-rich proteins, superoxide dismutase, and certain alternative mechanisms, has been described in the defence against oxidative stress. The role of the host-derived factors, particularly the phagocytosis of bacteria or host erythrocytes (intact or their enzymatic/non enzymatic components), in the detoxification of reactive oxygen metabolites in E. histolytica may provide novel approaches for the chemotherapy of invasive amoebiasis.

Interleukin-12 suppresses filaria-induced pulmonary eosinophilia, deposition of major basic protein and airway hyperresponsiveness.

Tropical Pulmonary Eosinophilia (TPE) is a severe form of allergic asthma caused by the host inflammatory response to filarial helminths in the lung microvasculature, and is characterized by pulmonary eosinophilia, increased filarial-specific IgG and IgE antibodies, and airway hyperresponsiveness. The current study examined the effect of IL-12 on pulmonary eosinophilia, deposition of eosinophil major basic protein and airway hyperresponsiveness in mice inoculated i.v. with Brugia malayi microfilariae. Injection of recombinant murine IL-12 modulated the T helper (Th) response in the lungs from Th2- to Th1-like, with elevated IFN-gamma, and decreased IL-4 and IL-5 production. Consistent with this shift in cytokine response, antigen-specific IgG2a was elevated, and IgG1 and total serum IgE were decreased. In addition, eosinophils in BAL fluid from IL-12 treated mice were reduced from 56% to 11%, and there was no detectable MBP on respiratory epithelial cells. Importantly, IL-12 suppressed airway hyperresponsiveness compared with saline-injected control animals. Taken together, these data clearly demonstrate that by modulating Th associated cytokine production, IL-12 down-regulates filaria-induced lung immunopathology.

An essential role for interleukin-5 and eosinophils in helminth-induced airway hyperresponsiveness.

Infection with the parasitic helminth Brugia malayi can result in development of a severe asthmatic response termed tropical pulmonary eosinophilia. This disease, thought to result from a host inflammatory response to blood parasites which become trapped in the lung microvasculature, is characterized by a profound eosinophilic infiltration into the lungs. Recruitment of eosinophils also correlates with the development of airway hyperresponsiveness (AHR) to cholinergic agonists and severe asthmatic symptoms. Our studies examined the role of interleukin-5 (IL-5) in helminth-induced pulmonary eosinophilia and AHR. C57BL/6 mice immunized with killed B. malayi microfilariae and challenged intravenously with live microfilariae exhibit many of the characteristics of human disease, including peripheral and pulmonary eosinophilia. Cells recovered by bronchoalveolar lavage of sensitized mice consisted of 3.8% eosinophils on day 1 postchallenge and 84% on day 10. Extracellular major basic protein was present on the surface of airway epithelial cells as early as day 1 and continued to be evident after 8 days, indicating sustained activation and degranulation of eosinophils in the lung. These histologic changes correlated with the development of AHR to carbachol. In contrast to immunocompetent mice, immunization and challenge with B. malayi in IL-5(-/-) mice did not induce peripheral or pulmonary eosinophilia, and these mice failed to show AHR in response to cholinergic agonists. Taken together, these data indicate that IL-5 and eosinophils are required for the induction of AHR by filarial helminths.

Differentiation of pathogenic and non-pathogenic Entamoeba: has the question been answered?

Pathogenic and non-pathogenic Entamoeba have been separated into two distinct species. Recently, the non-pathogenic E. dispar has been cultivated axenically. However, the genetic variability among different clones from the same strain, experimental production of hybrid clones which may differ from their parents, and the possibility of invasiveness of E. dispar, are some phenomena which may indicate that the last word on distinctiveness of the species has not yet been said.

Antioxidant defense mechanisms in parasitic protozoa.

Many of the parasitic protozoa, such as Entamoeba histolytica, Giardia, Trypanosoma, Leishmania, and Plasmodium, are considered to be anaerobes because they can be grown in vitro only under conditions of reduced oxygen tension. However, these parasitic protozoa have been found to be aerotolerant or microaerophilic, and also to consume oxygen to a certain extent. Furthermore, these organisms are highly susceptible to exogenous reactive oxygen species, such as hydrogen peroxide. They must, therefore, detoxify both oxygen and free radical products of enzymatic reactions. However, they lack some or all of the usual antioxidant defense mechanisms present in aerobic or other aerotolerant cells, such as catalase, superoxide dismutase, reduced glutathione, and the glutathione-recycling enzymes glutathione peroxidase and glutathione reductase. Instead, they possess alternative mechanisms for detoxification similar to those known to exist in certain prokaryotes. Although the functional aspects of these alternative mechanisms are yet to be understood completely, they could provide new insights into the biochemical peculiarities of these enigmatic pathogens.

In vitro susceptibility of Acanthamoeba culbertsoni to inhibitors of folate biosynthesis.

The effects of different sulphonamides, dihydrofolate reductase inhibitors and other inhibitors of folate metabolism on growth of Acanthamoeba culbertsoni in a chemically defined medium are reported. Among the sulphonamides, sulphamethoxazole and sulphadiazine were most effective followed by sulphanilamide and sulphaguanidine. Inhibition by each sulphonamide was reversed by p-aminobenzoic acid as well as folic acid. 7-Methylguanosine, a pteridine synthesis-inhibitor, did not inhibit multiplication of A. culbertsoni. Among the dihydrofolate reductase inhibitors, pyrimethamine blocked the amoebic growth at 100 micrograms/ml, while trimethoprim and cycloguanil palmoate failed to cause significant inhibition of growth even at 250 micrograms/ml. Metoprine inhibited amoebic growth completely at 50 micrograms/ml. Methotrexate and a thymidylate synthetase inhibitor 5-fluorouracil inhibited growth strongly, with IC50 values (the concentration of the drug which causes 50% inhibition of the growth at 72 h) of 1.97 and 2.45 micrograms/ml, respectively. Inhibition by methotrexate, metoprine or 5-fluorouracil could not be reversed by folic acid, folinic acid, thymidine, or folinic acid plus thymidine. The results indicate unusual features in A. culbertsoni folate metabolism.

Nutritional studies on Acanthamoeba culbertsoni and development of chemically defined medium.

A chemically defined medium containing 11 amino acids, 3 vitamins, 6 inorganic salts and glucose, yielding maximum cell densities of 1.5-2.5 x 10(7) cells/ml, has been developed for Acanthamoeba culbertsoni with a mean generation time (MGT) of 10 h. A medium containing six amino acids viz. arginine, methionine, leucine, isoleucine, valine and glycine along with other components could also support good albeit slower growth (MGT 27 h) of the amoeba. Acetate did not serve as a suitable carbon/energy source for A. culbertsoni. This organism bears close resemblance in its nutritional requirements to other Acanthamoeba especially A. polyphaga.

Development of improved media for axenic cultivation of Acanthamoeba culbertsoni, Singh and Das 1970.

Several varieties of peptone supported growth of A. culbertsoni to different extents reaching a maximum cell density of 1-2 X 10(6)/ml. Proteose peptone and tryptone also yielded good growth when combined with thiamine and vitamin B12. A combination of proteose peptone with glucose, yeast extract and salts promoted excellent growth of A. culbertsoni with cell density reaching 1-2 X 10(7) cells/ml; tryptone and one of the indigenous peptones also yielded comparable growth when substituted for proteose peptone in this medium. Casamino acids also supported good growth of amoebae and requirement of yeast extract could be met by a combination of thiamine, vitamin B12 and biotin. Bacto peptone did not support good growth of this amoeba but supplementation of peptone with casamino acids or amino acid mixture improved the growth supporting capacity of the medium. Development of several media with or without glucose will aid in cultivation of A. culbertsoni, studies on its metabolism as well as screening of potential drugs.

Reducing agents and Entamoeba histolytica.

Entamoeba histolytica remains an important but enigmatic parasite. It displays both non-pathogenic and invasive pathogenic types, which can be distinguished clinically and by isoenzyme markers. Yet as debated in Parasitology Today last year(1), the relationship between these two forms remains unclear. Bacterial associates and reducing agents are known to play on important role in the culture of E. histolytica, and possibly in its differentiation and invasive mechanisms. This article briefly reviews available information on the role o f reducing agents, and explores the possibility that bacteria may play a role in reduction o f toxic oxygen product - thereby promoting the virulence of E. histolytica. The review is not definitive, but should help to stimulate further research in this neglected area.