Hossein Keshavarz

CD8+ T cells as a source of IFN-γ production in human cutaneous leishmaniasis.

Background In human leishmaniasis Th1/Th2 dichotomy similar to murine model is not clearly defined and surrogate marker(s) of protection is not yet known. In this study, Th1/Th2 cytokines (IL-5, IL-10, IL-13 and IFN-γ) profile induced by purified CD4+/CD8+ T cells in response to Leishmania antigens were assessed at transcript and protein levels in 14 volunteers with a history of self-healing cutaneous leishmaniasis (HCL) and compared with 18 healthy control volunteers. Methodology/Principal Findings CD4+/CD8+/CD14+ cells were purified from peripheral blood using magnetic beads; CD4+/CD8+ T cells were co-cultured with autologous CD14+ monocytes in the presence of soluble Leishmania antigens (SLA). Stimulation of either CD4+ T cells or CD8+ T cells of HCL volunteers with SLA induced a significantly (P<0.05) higher IFN-γ production compared with the cells of controls. Upregulation of IFN-γ gene expression in CD4+ cells (P<0.001) and CD8+ cells (P = 0.006) of HCL volunteers was significantly more than that of controls. Significantly (P<0.05) higher fold-expression of IFN-γ gene was seen in CD4+ cells than in CD8+ cells. In HCL volunteers a significantly (P = 0.014) higher number of CD4+ cells were positive for intracellular IFN-γ production than CD8+ cells. Conclusions/Significance Collectively, the volunteers have shown maintenance of specific long-term immune responses characterized by a strong reaction to leishmanin skin test and IFN-γ production. The dominant IFN-γ response was the result of expansion of both CD4+ and CD8+ T cells. The results suggested that immune response in protected individuals with a history of zoonotic cutaneous leishmaniasis (ZCL) due to L. major is mediated not only through the expansion of antigen-specific IFN-γ producing CD4+ Th1 cells, but also through IFN-γ producing CD8+ T cells.

Rapid detection of human and canine visceral leishmaniasis: Assessment of a latex agglutination test based on the A2 antigen from amastigote forms of Leishmania infantum

The diagnosis of visceral leishmaniasis (VL) in humans and animal reservoir hosts is difficult, particularly in rural areas where the disease is endemic and laboratory facilities are limited. This study aimed to develop a latex agglutination test (LAT) for the rapid detection of anti-Leishmania antibodies against the A2 antigen derived from the amastigote form as well as those against crude antigens derived from the promastigote form of an Iranian strain of Leishmania (Leishmania) infantum. The A2 antigen (42-100 kDa) was prepared from the amastigote form of L. infantum, purified with electroelution and compared with the crude antigen from the promastigote form of L. infantum. Both antigens showed appropriate intensity reactions, were selected using dot blotting of positive and negative pooled sera and used to sensitize 0.9-μm latex beads. The tests were carried out on sera from 43 symptomatic, human patients with VL confirmed by parasitological examination and direct agglutination test (DAT), 30 healthy controls and 32 patients with other infections but without VL. Canine sera were collected from 63 domestic dogs with VL confirmed using parasitological examinations and DAT and 31 healthy dogs from areas non-endemic for VL. Compared with the controls, human sera from DAT-confirmed patients yielded a sensitivity of 88.4% (95% CI, 82.1-94.5%) and specificity of 93.5% (95% CI, 87.0-99.7%) on A2-LAT (amastigote) when 1:3200 was used as the cut-off titre. A good degree of agreement was found between A2-LAT and DAT (0.914). LAT required 3-5 min to complete, versus the 12-18 h needed for DAT. Compared with the controls, A2-LAT of canine sera from DAT-confirmed cases yielded a sensitivity of 95.2% (95% CI, 95.0-95.4%) and specificity of 100% (95% CI 100%) when 1:320 was used as the cut-off titre. A good degree of agreement was found between A2-LAT and DAT (0.968). Similarly, the sensitivity and specificity of Pro.-LAT (promastigote) was calculated to be 88.4% and 91.9%, respectively for human sera and 96.8% and 90.3%, respectively for canine sera. No statistically significant differences were observed between A2-LAT and Pro.-LAT for the detection of human and canine L. infantum infections. In conclusion, A2-LAT and Pro.-LAT could be used in parallel to screen for L. infantum infections in humans and dogs in areas endemic for VL in Iran.

DO MUTATIONS IN PLASMODIUM FALCIPARUM DIHYDROPTEROATE SYNTHASE AND DIHYDROFOLATE REDUCTASE CONFER RESISTANCE TO SULFADOXINE-PYRIMETHAMINE IN IRAN?

The key codons of dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) genes implicated in sulfadoxine-pyrimethamine (SP) resistance were determined by allele-specific polymerase chain reaction in Plasmodium falciparum isolates collected in 1999 from 35 Iranian patients treated with SP. Seven isolates had Glu-540 dhps allele but 5 of these isolates were characterized to possess wild-type dhfr alleles. Seven additional isolates were polyclonal with mixed Lys- and Glu-540. The key dhfr mutation associated with pyrimethamine resistance, Asn-108, was found in 4 isolates. In one patient the presence of Lys- and Glu-540 in dhps and Asn-108 and Arg-59 in dhfr was associated with treatment failure. However, more studies are needed to determine whether clinical response to SP and mutations in these genes are correlated in Iran.

Production and Evaluation of Toxoplasma gondii Recombinant GRA7 for Serodiagnosis of Human Infections

The precise diagnosis of the acute toxoplasmosis in pregnant women and immunocompromsied patients has critical importance. Most of the commercially available assays use the whole Toxoplasma soluble extract as the antigen. However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the lack of a purified standardized antigen. The aim of this study was production and evaluation of the usefulness of the recombinant Toxoplasma gondii GRA7 antigen for the serodiagnosis of Toxoplasma gondii IgM and IgG by ELISA. A total of 70 T. gondii IgM positive sera, 74 T. gondii IgG positive sera, and 60 sera from subjects who were not infected with T. gondii were examined. These sera were shown different absorbance values in ELISA test. To control the specificity of the rGRA7 other parasitic diseases, for example, echinococcosis, malaria, leishmaniasis, fascioliasis, and strongyloidiasis were tested of which none showed positive results. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (com ELISA) were 89% and 90%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 96% and 90%, respectively. The results obtained here show that this antigen is useful for diagnostic purposes.

Genotypes and in vivo resistance of Plasmodium falciparum isolates in an endemic region of Iran.

Mutations in the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum have been correlated with and used to detect antifolate treatment failure, such as sulfadoxine–pyrimethamine (SP), in regions endemic for malaria. To determine the association between molecular markers of SP resistance and in vivo drug resistance, a quick and simple technique that detects single nucleotide polymorphisms in the DHFR and DHPS genes, using PCR–ELISA and sequence-specific oligonucleotide probes, was applied to 53 isolates obtained from an in vivo study in Sistan and Baluchistan Province, in southeastern Iran. Overall, 11.3% of these isolates were obtained from patients with SP treatment failure. Four DHFR polymorphisms (codons 51, 59, 108, and 164) and five DHPS polymorphisms (codons 436, 437, 540, 581, and 613) were investigated. Mutations DHFR Asn-108, DHFR Arg-59, and DHPS 436-Ala/Phe were very common (100, 81.1, and 85%, respectively). Plasmodium falciparum was isolated from 96% of patients with at least two DHFR/DHPS mutations. All resistant isolates had at least three mutations. The high prevalence of mutation associated with antifolate resistance may point toward low drug efficacy in the future.

Monitoring of the therapeutic efficacy of chloroquine for the treatment of uncomplicated, Plasmodium falciparum malaria in Iran

Abstract Between 2002 and 2004, the standardized 28-day protocol recently developed by the World Health Organization was used to explore the efficacy of chloroquine, in the treatment of uncomplicated, Plasmodium falciparum malaria, in five sentinel sites in southern Iran. All but 14 of the 158 patients enrolled (128, 28 and two from the provinces of Sistan–Baluchestan, Hormozgan and Kerman, respectively) were successfully followed-up. The overall frequency of treatment failure by day 28 was 78.5%, with 17.4% of the patients being classed as early treatment failures, 34.7% as late clinical failures, and 26.4% as late parasitological failures. There appeared to be no significant change in the frequency of treatment failure between the 2002–2003 and 2003–2004 transmission seasons, nor any significant between-site variation in the efficacy of chloroquine. Given these observations, the replacement of chloroquine, as the first-line drug for the treatment of uncomplicated, P. falciparum malaria in Iran, was inevitable. Artesunate–sulfadoxine–pyrimethamine is now the recommended first–line treatment, with artemether–lumefantrine used for second-line treatment. The efficacies of these combination therapies are currently being evaluated and monitored.

Genetic diversity and natural selection at the domain I of apical membrane antigen-1 (AMA-1) of Plasmodium falciparum in isolates from Iran

The apical membrane antigen-1 (AMA-1) of Plasmodium falciparum is a prime malaria asexual blood-stage vaccine candidate. Antigenic variation is one of the main obstacles in the development of a universal effective malaria vaccine. The extracellular region of P. falciparum AMA-1 (PfAMA-1) consists of three domains (I-III), of which the domain I is the most diverse region of this antigen. The objective of our study was to investigate and analyze the extent of genetic diversity and the effectiveness of natural selection at the AMA-1 domain I of P. falciparum in isolates from Iran. A fragment of ama-1 gene spanning domain I was amplified by nested PCR from 48 P. falciparum isolates collected from two major malaria endemic areas of Iran during 2009 to August 2010 and sequenced. Genetic polymorphism and statistical analyses were performed using DnaSP and MEGA software packages. Analysis of intrapopulation diversity revealed relatively high nucleotide and haplotype diversity at the PfAMA-1 domain I of Iranian isolates. Neutrality tests provided strong evidence of positive natural selection acting on the sequenced gene region. The findings also demonstrated that, in addition to natural selection, intragenic recombination may contribute to the diversity observed at the domain I. The results obtained will have significant implications in the design and the development of an AMA-1-based vaccine against falciparum malaria.

Detection of parasitic particles in domestic and urban wastewaters and assessment of removal efficiency of treatment plants in Tehran, Iran

BackgroundIn recent years, decreasing annual rainfalls in some countries and population growth have led to a shortage of freshwater resources. Thus, recycled wastewaters has been suggested for agricultural activities. Contamination of wastewaters with pathogens is a major concern for the use of these waters. This study aimed to (i) investigate the occurrence of helminth eggs and protozoan (oo)cysts in human and livestock wastewaters, and (ii) evaluate the parasite removal efficiencies of urban and slaughterhouse treatment plants in Tehran province, Iran. One hundred and eight samples were collected from five urban and domestic wastewater treatment plants in Iran. Wastewater samples were concentrated by centrifugal-concentration and filtration methods.ResultsThe quantity of helminths egg and protozoa (oo)cyst per liter of urban raw wastewater ranged from 1.2 × 101 to 2.9 × 101 and from 9.6 × 102 to 1.9 × 103, respectively. The number of eggs and (oo)cysts per liter of animal raw wastewater ranged from 1.6 × 103 to 4.9 × 103 and 3.1 × 104 to 6.0 × 104, respectively. The helminths and protozoa identified in urban treatment plants included hookworms, Hymenolepis and Rhabditis (or probably Strongyloides), Entamoeba, Isospora, Giardia, Chilomastix and Cryptosporidium, while in slaughterhouses Trichuris, Trichostrongylus, Moniezia, Dicrocoelium, Fasciola, Entamoeba, Cryptosporidium, Eimeria and Giardia were isolated. The overall removal efficiency of eggs and (oo)cysts in the treatment plants ranged from 94.8 to 95.7% and from 79.3 to 85.8%, respectively.ConclusionThe study results revealed that the efficacy of removal of nematode eggs, and not protozoan (oo)cysts, in urban wastewater treatment plants, is in compliance with the WHO parasitological guideline (<1 nematode per liter) required for unrestricted irrigation.

The possible association between Toxoplasma gondii infection and risk of anxiety and cognitive disorders in BALB/c mice

There are conflicting reports concerning the association of Toxoplasma gondii infection with increased risk of mental disorders. This investigation will provide a good understanding about defining the possible association between T. gondii exposure and risk of anxiety and cognitive alterations. Besides, a secondary objective of this study was to determine the effect of pioglitazone administration on the possible alterations induced by T. gondii exposure. Male BALB/c mice were used for this study. The animal model of Toxoplasma infection was established by the intraperitoneal inoculation of 20–25 tissue cysts from Tehran strain of T. gondii. Pioglitazone (20 mg/kg, i.p.1/day) was administered to the animals for 2 weeks before behavioural tests. Behavioural tests including open-field, elevated plus-maze and passive avoidance learning were evaluated in the groups. Since cytokines were implicated as a contributing factor for mood disorders, the mRNA levels of TNF-α, IL-1β, IL-6 as well as inducible nitric oxide synthase (iNOs) were examined by real-time PCR. Findings demonstrated that T. gondii caused anxiety-like symptoms and impaired cognitive functions of the infected BALB/c mice, whereas pioglitazone, a peroxisome proliferator-activated receptor agonist, showed a promising effect against the cognitive impairments induced by Toxoplasma infection. The results also revealed that the mRNA levels of the aforementioned cytokines were significantly (p < 0.05) increased in the infected mice compared to the uninfected BALB/c ones. Pioglitazone can be offered as a potential neuroprotective agent in the treatment of patients with T. gondii infection that manifests anxiety and cognitive impairments; however, further studies are needed to clarify the exact mechanisms.

T Helper 1 (Th1), Th2, and Th17 Responses to Leishmania major Lipophosphoglycan 3

ABSTRACT Leishmania major is the main causal agent of cutaneous leishmaniasis (CL) that remains a serious public health concern in many tropical and subtropical countries. A long-lasting protective vaccine against leishmaniasis remains as a medical unmet need. Lipophosphoglycan 3 (LPG3) is one of the class II LPG genes from HSP90 family involved in the host immune responses. The aim of the present study is to investigate the capability of recombinant LPG3 (rLPG3) to induce Th1, Th2, Th17 responses. The results showed that rLPG3 in moderate and high concentrations significantly induced expression of Th1 lineage-specific transcription factor (T-bet) and cytokine (IFN-γ)(P < 0.05). Moreover, the Th1-stimulating effect of rLPG3 was confirmed by significant induction of IFN-γ secretion from treated T cells (P < 0.01). However, no significant effect of rLPG3 on Th2 and Th17 lineage cells was observed even in high concentration. Our findings demonstrate that rLPG3 induces Th1, but not Th2 and Th17, lineage responses. Further studies are needed to investigate adjuvant properties of rLPG3 for leishmania therapy.