Comparison of pro-inflammatory cytokines of non-healing and healing cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) heals spontaneously within several weeks or months, but, in rare cases, CL-active lesions last for many years. In this study, we assessed cell-mediated immunity in non-healing CL through the measurement of three pro-inflammatory cytokines: Interferon-γ (IFN-γ), IL-17a and CXCL-11. For this, 32 patients afflicted with healing or non-healing CL were recruited in this study. Peripheral blood mononuclear cells (PBMCs) of every patient were treated with three antigens: purified protein derivative (PPD), soluble Leishmania antigen (SLA) and phytohaemagglutinin (PHA). Cytokine quantification was performed using enzyme-linked immunosorbent assay (ELISA) method. Results of our study showed that neither cytokine produced in the presence of a PPD stimulator (as an irrelevant antigen) significantly differed between the healing and non-healing groups (P-value ≥0.05 for all of them). However, IFN-γ, CXCL-11 and IL-17a levels produced in the presence of PHA or SLA were significantly higher within the healing than in the non-healing group (P-value <0.01 for all of them). It seems that appropriate levels of IFN-γ, as well as IL-17a and CXCL-11, contribute to the control of Leishmania infection.

Immunogenicity of HLA-DR1 Restricted Peptides Derived from Leishmania major gp63 Using FVB/N-DR1 Transgenic Mouse Model.

BACKGROUND: Leishmaniasis is a worldwide disease prevalent in tropical and sub-tropical countries. In the present study the immunogenicity of three human HLA-DR1 restricted peptides derived from L. major gp63 protein was evaluated using FVB/N-DR1 transgenic mouse model. METHODS: The immunity generated by three MHC class II - restricted peptides with the sequence of AARLVRLAAAGAAVT (AAR), AAPLVRLAAAGAAVT (AAP) and SRYDQLVTRVVTHE (ASR) derived from L. major gp63 protein were predicted using a web-based software (SYFPEITHI) and tested in FVB/N-DR1 transgenic mice. RESULTS: Immunization of FVB/N-DR1 transgenic mice with one of the three predicted peptides (AAR) resulted in high levels of Th1-type immune response as well as significant levels of IFN-γ detected by Proliferation assay and ELISA. CONCLUSION: The results indicate a high level of immunogenicity for AAR, which can be a potent candidate for peptide vaccine in Leishmania infections.

Association of BMPR-1B and GDF9 genes polymorphisms and secondary protein structure changes with reproduction traits in Mehraban ewes.

BMPR-1B and GDF9 genes are well known due to their important effects on litter size and mechanisms controlling ovulation rate in sheep. In the present study, polymorphisms of BMPR-1B gene exon 8 and GDF9 gene exon 1 were detected by single strand conformational polymorphism (SSCP) analysis and DNA sequencing methods in 100 Mehraban ewes. The PCR reaction forced to amplify 140 and 380-bp fragments of BMPR-1B and GDF9 genes, respectively. Two single nucleotide polymorphisms (SNPS) were identified in two different SSCP patterns of BMPR-1B gene (CC and CA genotypes) that deduced one amino acid exchange. Also, two SNPS were identified in three different SSCP patterns of GDF9 gene (AA, AG and GG genotypes) that deduced one amino acid exchanges. Two different secondary structures of protein were predicted for BMPR-1B exon 8, but the secondary protein structures predicted for GDF9 exon 1 were similar together. The evaluation of the associations between the SSCP patterns and the protein structure changes with reproduction traits showed that BMPR-1B exon 8 genotypes have significant effects on some of reproduction traits but the GDF9 genotypes did not have any significant effect. The CA genotype of BMPR-1B exon 8 had a significant positive effect on reproduction performance and could be considered as an important and new mutation, affecting the ewes reproduction performance. Marker assisted selection using BMPR-IB gene could be noticed to improve the reproduction traits in Mehraban sheep.

Molecular beacon probes-base multiplex NASBA Real-time for detection of HIV-1 and HCV.

BACKGROUND AND OBJECTIVES: Developed in 1991, nucleic acid sequence-based amplification (NASBA) has been introduced as a rapid molecular diagnostic technique, where it has been shown to give quicker results than PCR, and it can also be more sensitive. This paper describes the development of a molecular beacon-based multiplex NASBA assay for simultaneous detection of HIV-1 and HCV in plasma samples. MATERIALS AND METHODS: A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used for primers and molecular beacon design. The performance features of HCV/HIV-1 multiplex NASBA assay including analytical sensitivity and specificity, clinical sensitivity and clinical specificity were evaluated. RESULTS: The analysis of scalar concentrations of the samples indicated that the limit of quantification of the assay was <1000 copies/ml for HIV-1 and <500 copies/ml for HCV with 95% confidence interval. Multiplex NASBA assay showed a 98% sensitivity and 100% specificity. The analytical specificity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes detected all HCV genotypes and all major variants of HIV-1. CONCLUSION: This method may represent a relatively inexpensive isothermal method for detection of HIV-1/HCV co-infection in monitoring of patients.

Immunogenicity of MHC Class I Peptides Derived from Leishmania mexicana Gp63 in HLA-A2.1 Transgenic (HHDII) and BALB/C Mouse Models.

BACKGROUND: Leishmania is an intracellular parasite infecting humans and many wild and domestic animals. Recent studies have suggested an important role for cytotoxic T cells against Leishmania. Peptide-based vaccines targeting short sequences derived from known immunogenic proteins have been shown to elicit cellular immune responses against disparate pathogens. METHODS: We predicted four HLA-A2 peptides derived from L. mexican/major gp63 and tested these in HHD II mice, as well as four peptides for mouse MHC class I from the same proteins tested in BALB/ mice. RESULTS: The results revealed immunogenicity for three of the four peptides predicted for HLA-A2. Immunisation with these peptides, along with IFA, induced CTL responses detected by standard 4-hour cytotoxicity assay and significantly upregulated the production of IFN-γ. When HHDII mice were injected IM with L. mexicana gp63 cDNA and splenocytes were restimulated with blasts loaded with the immunogenic peptides, two of the peptides were able to induce significant level of IFN-γ detected by ELISA. None of the peptides predicted for Balb/c mouse MHC class I elicited CTL activity or significantly upregulated the IFN-γ. CONCLUSION: The results may help in developing a peptide-based vaccine, which can be applied alone or in combination with drugs against Leishmania.

Leishmania mexicana Gp63 cDNA Using Gene Gun Induced Higher Immunity to L. mexicana Infection Compared to Soluble Leishmania Antigen in BALB/C.

BACKGROUND: Leishmaniasis is a worldwide disease prevalent in tropical and sub tropical countries. Many attempts have been made and different strategies have been approached to develop a potent vaccine against Leishmania. DNA immunisation is a method, which is shown to be effective in Leishmania vaccination. Leishmania Soluble Antigen (SLA) has also recently been used Leishmania vaccination. METHODS: The immunity generated by SLA and L. mexicana gp63 cDNA was compared in groups of 6 mice, which were statistically analysed by student t- test with the P-value of 0.05. SLA was administered by two different methods; intramuscular injection and injection of dendritic cells (DCs) loaded with SLA. L. mexicana gp63 cDNA was administered by the gene gun. RESULTS: Immunisation of BALB/c mice with L. mexicana gp63 resulted in high levels of Th1-type immune response and cytotoxic T lymphocytes (CTL) activity, which were accompanied with protection induced by the immunisation against L. mexicana infection. In contrast, administration of SLA, produced a mixed Th1/Th2-type immune responses as well as a high level of CTL activity but did not protect mice from the infection. CONCLUSION: The results indicate higher protection by DNA immunisation using L. mexicana gp63 cDNA compared to SLA, which is accompanied by a high level of Th1 immune response. However, the CTL activity does not necessarily correlate with the protection induced by the vaccine. Also, gene gun immunisation is a potential approach in Leishmania vaccination. These findings would be helpful in opening new windows in Leishmania vaccine research.

Prevalence of Nonalcoholic Steatohepatitis in Iran: A Population based Study.

BACKGROUND Non-alcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) is the most common form of chronic liver disease worldwide and is no longer considered a benign disease. Its prevalence has not been determined in a large-scale population-based study in Iran. METHODS A total of 6583 individuals aged 18 to 65 were randomly selected from three geographically distinct provinces in Iran. Blood samples were obtained from each subject and a questionnaire was completed exploring data including self-admitted regular alcohol use. Serums were tested for anti-HCV antibody (anti-HCV), hepatitis B surface antigen and anti-hepatitis B core antibody. Positive samples for anti-HCV antibody were re-tested and those positive in a repeat ELISA were confirmed by a recombinant immunoblot assay (RIBA) test. Serums were also tested for ALT levels. Subjects with elevated ALT defined as serum ALT ≥40 IU/L with no history of alcohol consumption and negative HBV and HCV infection were considered as "presumed NASH". RESULTS In this study 5589 subjects were analyzed. Two hundred and forty two individuals (4.3%) were diagnosed with elevated ALT levels. Among individuals with elevated ALT, 15 (6.2%) were diagnosed with either hepatitis B or hepatitis C. The overall weighted prevalence of presumed NASH was 2.9%. According to multivariate analysis, male sex, urban lifestyle, and being overweight or obese were significantly associated with "presumed NASH". CONCLUSION Obesity and metabolic syndrome, the most predictive factors of fatty liver disease, are increasing in Iran, therefore the prevalence of NAFLD/NASH and related complications are expected to increase in the future. This population based study gives a crude estimate of the prevalence of NASH around the country. Studies with more accurate surrogates of NASH need to be done. The disparity among different provinces merits special consideration.

A Population-based Seroepidemiological Study on Hepatitis E Virus in Iran.

BACKGROUND Little is known about HEV seroprevalence and its determinants in Iran. Considering the fact that Iran is among the countries in which HEV infection is endemic, a large-scale population-based study in this regard is justified. METHODS This survey was conducted in 2006 in Tehran and Golestan Provinces, Iran. Stored sera of subjects were tested for serological markers of anti-HEV. The baseline data were recorded in structured questionnaires. Weighted seroprevalence and weighted logistic regression coefficients were calculated. RESULTS A total of 1423 samples were included. The overall seroprevalence in two provinces was 7.4%. Age with an odds ratio equal to 1.59 (95% CI: 1.26-2.02) and history of traditional phlebotomy with an odds ratio equal to 2.28 (95% CI: 1.13-4.60) were independent predictors of HEV seropositivity. CONCLUSION Considering the high rate of HEV seroprevalence in Iran, further studies on the cost-effectiveness of vaccination among vulnerable groups are mandatory.

Trends in prevalence of hepatitis B virus infection among Iranian blood donors, 1998-2007.

Assessment of the quality of donor selection and safety of the blood supply can be estimated by monitoring the prevalence of the serologic markers of infectious disease in screening tests. In the present study, changes in rates of hepatitis B virus (HBV) infection are studied in the period 1998-2007 in Iranian Blood Transfusion Organization (IBTO). Prevalence of serological marker of HBV infection [hepatitis B surface antigen (HBsAg)] was evaluated in blood donations in Iran as well as for Fars province representing a low prevalence, and Sistan-Baluchestan (S&B) province as a high prevalence region throughout 1998-2007. For assessing frequency of infection, the prevalence of HBsAg per 100 000 donations and 95% confidential intervals (95% CIs) is calculated. P value is estimated by chi(2) test. A total of 14 599 783 donations were collected during 10 years. The overall HBsAg prevalence rates declined from a 1.79% (1789/100 000 donations) in 1998 to 0.41% (409/100 000 donations) in 2007 in Iran. In Fars province, HBsAg prevalence decreased from 0.89% in 92 999 donations in 1998 to 0.34% in 148 014 donations in 2007 and in S&B province, the rate of HBsAg has gone down from 3.74% in 44 036 donations in 1998 to 1.15% in 56 057 donations in 2007. The frequency of HBV infection entering the blood supply has decreased over this period as a result of improvement in donor recruitment and selection, usage of software in transfusion services and possibly decreasing HBV infection prevalence in general population.

CTL responses to Leishmania mexicana gp63-cDNA vaccine in a murine model.

Immunity to Leishmania is believed to be strongly dependent upon the activation of Th1 immune responses, although the exact role of cytotoxic T lymphocytes (CTLs) has not yet been determined. The aims of this study were to establish a suitable cytotoxicity assay to measure CTL activity and to compare immunity induced by Leishmania mexicana gp63 cDNA via i.m. injection and gene gun immunization in the BALB/c mouse model. The CTL activity was evaluated by short-term (51)Cr-release cytotoxicity assays against CT26 tumour cells transfected with L. mexicana gp63 cDNA and dendritic cells (DCs) loaded with soluble Leishmania antigen (SLA) as targets. The results clearly demonstrated that higher protection to L. mexicana infection was induced by gene gun DNA-immunization vs. i.m. injection. Cytotoxic T lymphocyte activity of splenocytes was observed in mice immunized either with L. mexicana gp63 cDNA or SLA and long-lived CTL activity was observed in immunized and/or re-challenged mice but not naïve mice infected with the parasite.

Transfusion-transmitted infections among multitransfused patients in Iran: a review.

Transfusion-transmitted infections (TTI) continue to be a major challenge for Blood transfusion organizations across the world. The problem is more serious in the developing countries with lower economic means. Multitransfused patients (MTPs) in these countries are at higher risk of infection, and studies of infection in these patients can be a useful index for examining the blood safety filters in place. The present article reviews the situation in Iran, where prevalence of the major viruses of concern, namely, hepatitis B virus, hepatitis C virus (HCV) and human immunodeficiency virus, studied in these patients is reported over a 9-year period. It is demonstrated that HCV is the most prevalent TTI and remains a major health problem for these patients.

Molecular and serological characterization of hepatitis B virus in deferred Ghanaian blood donors with and without elevated alanine aminotransferase.

Candidate blood donors in Ghana are frequent carriers of hepatitis B virus (HBV). A comparative study of 117 donor samples including 46 with alanine aminotransferase (ALT) > or = 60 IU/L and 71 with < or =40 IU/L level was undertaken. S and the basic core promoter-precore regions (BCP/PC) sequencing was used to identify genotypes and variants relevant to HBV natural history, respectively. Age, viral load, HBe status were correlated with molecular data. HBV genotype E (87%) was dominant with little genotypes A (10%) and D (3%). Comparing individuals with or without liver disease, an association between liver disease and older age (P = 0.004) and higher viral load (P = 0.002) whether as a whole population or only genotype E was found. Compared with a commercial assay, BCP/PC sequencing had lower sensitivity to detect mixtures of wild-type and variant viruses but detected BCP deletions. BCP 1762/1764 variants were positively correlated with older age (P < 0.0001) and elevated ALT levels (P = 0.01). PC 1896 stop codon was marginally correlated with viral load (P = 0.09). HBV genotype E infection natural history appears different from genotypes B and C prevalent in Asia. Donors with liver disease being older, with higher viral load and higher BCP variant proportion may be at higher risk of cirrhosis and hepatocellular carcinoma.

A study on delta virus infection and its clinical impact in Iran.

During the period 1986-1988, the expression of anti-HDV in different high-risk groups and its clinical impact on patients with HBV-related chronic liver disease and hepatocellular carcinoma was investigated in Iran. Using the ELISA technique, we observed a 2.5% anti-HDV positivity in asymptomatic chronic HBsAg carriers (3 of 120); in hemophiliacs, two of six HBsAg carriers were positive for anti-HDV and zero of 50 anti-HBs positives. Anti-HBs positive dialysis patients were positive for anti-HDV in 2.0% of the cases (1 of 50), whereas the rate of anti-HDV positivity was 44.5% in hemodialysis patients positive for HBsAg (16 of 36). The figures were comparable in HBsAg positive patients with chronic active hepatitis and cirrhosis (49.2%; 31 of 63). Moreover, anti-HDV was detected in five of eight patients with hepatocellular carcinoma. These data indicate the endemicity of delta infection in Iran. The increased incidence among hepatocellular carcinoma patients is an interesting finding to be further investigated with larger groups of patients in this region.

Circular dichroic, infrared and other studies on the protein component of pig brain thromboplastin.

1. A four-step procedure used to isolate the protein component (apoprotein III) of pig brain thromboplastin yielded approximately 25 mg from 500g of brain. 2. In the absence of detergent, apoprotein III had an apparent mol.wt. of 360 000 by gel-filtration, and, after electrophoresis on polyacrylamide gels in the presence of sodium docecyl sulphate, it appeared as a major protein band of mol.wt.59 000, suggesting the existence of polymeric and monomeric forms. 3. Chemical analyses of apoprotein III revealed that hydrophilic and hydrophobic amino acids were present in a ratio of 3:2, together with approx, 9% (w/w) of carbohydrate. 4. The far-u.v.c.d. and i.r. spectral data indicated that, like other membrane proteins, apoprotein III has a high percentage of unordered structure with lesser amounts of alpha and beta-forms. 5. Relipidation of apoprotein III to restore clotting activity caused no extensive alteration in the c.d. and i.r. spectra, indicating that the phospholipid associates with a comparatively small hydrophobic segment. The constrained unordered conformation, which makes the major contribution to the c.d. spectrum, probably forms a separate domain in the aqueous phase. The absence of any increase in the amplitude of both negative c.d. extrema, following relipidation, contrasted with the substantial increase observed in a helix-forming solvent and raises the possibility that the more stable polymeric form of apoprotein III is retained as the active form in the lipid phase. 6. We suggest that as a consequence of cell membrane damage, the recognition and activation of factor VII may involve minor changes of conformation that are dependent upon the flexibility inherent in an unordered secondary structure.