Heat Shock Proteins Enriched-Promastigotes of Leishmania major Inducing Th2 Immune Response in BALB/c Mice

Background

Heat shock proteins (HSP) are highly conserved molecules with many immunological functions. They are highly immunogenic with important role in cancer immunotherapy and in vaccine development against infectious diseases. As adjuvant, HSP can augment the immunogenicity of weak antigens and can stimulate antigen presenting cells. Although vaccines have been successful for many infectious diseases, progress in leishmaniasis has not been achieved. In this report, the protective effect of HSP-enriched soluble leishmania antigen (SLA) was determined.

Methods

BALB/c mice were immunized 3× with HSP-enriched SLA and SLA alone and 10 days after final boost. They were infected with 10⁶ stationary phase promastigote of Leishmania major and immunological responses were followed until nine weeks.

Results

No significant differences were observed in lymphocyte proliferation, footpad swelling, parasite burden, nitric oxide or IL-12 cytokine between HSP-enriched or SLA groups. Although the levels of IFN-γ, IL-4, TGF-β, IgG1 and IgG2b were increased in both groups, IFN-γ was significantly higher in SLA group and IgG2a in HSP-enriched SLA.

Conclusion

These results indicate that HSP direct the immune system towards Th2 pattern and does not have protective role in L. major infection. Key Words: Leishmaniasis, Heat shock proteins (HSP), Adjuvant     

Design of a Novel Recombinant Multi-Epitope Vaccine against Triple-Negative Breast Cancer

Background:TNBC is determined by the absence of ERBB2, estrogen and progesterone receptors’ expression. Cancer vaccines, as the novel immunotherapy strategies, have emerged as promising tools for treating the advanced stage of TNBC. The aim of this study was to evaluate CEA, MTDH, and MUC-1 proteins as vaccine candidates against TNBC. Methods:In this research, a novel vaccine was designed against TNBC by using different immunoinformatics and bioinformatics approaches. Effective immunodominant epitopes were chosen from three antigenic proteins, namely CEA, MTDH, and MUC-1. Recombinant TLR4 agonists were utilized as an adjuvant to stimulate immune responses. Following the selection of antigens and adjuvants, appropriate linkers were chosen to generate the final recombinant protein. To achieve an excellent 3D model, the best predicted 3D model was required to be refined and validated. To demonstrate whether the vaccine/TLR4 complex is stable or not, we performed docking analysis and dynamic molecular simulation. Result:Immunoinformatics and bioinformatics evaluations of the designed construct demonstrated that this vaccine candidate could effectively be used as a therapeutic armament against TNBC. Conclusion:Bioinformatics studies revealed that the designed vaccine has an acceptable quality. Investigating the effectiveness of this vaccine can be confirmed by supplementary in vitro and in vivo studies.Key Words: Adjuvants, Triple-negative breast cancer, Vaccine     

Immunogenicity Evaluation of a DNA Vaccine Expressing the Hepatitis C Virus Non-Structural Protein 2 Gene in C57BL/6 Mice

Backgrounds: Most of the hepatitis C virus (HCV) infections elicit poor immune responses and 75% to 85% of cases become chronic; therefore, the development of an effective vaccine against HCV is of paramount importance. In this study, we aimed to evaluate co-administration of HCV non-Structural Protein 2 and IL-12 DNA vaccines in C57BL/6 mice. Methods: A plasmid encoding full-length HCV NS2 protein (non-structural protein 2) was generated and used to vaccinate mice. Negative control (an empty expression vector) was also employed to evaluate the background response. To investigate immune responses against vaccine, C57BL/6 mice received three doses of the vaccine with a two-week interval. Cellular immunity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay for lymphocyte proliferation, lactate dehydrogenase release for cytotoxic T lymphocyte (CTL) activity and cytokine assay. Results: The findings demonstrated that immunization of mice with plasmid expressing HCV NS2 induced CTL response, interferon gamma production, and lymphocyte proliferation compared to negative control. The results also demonstrated that co-administration of IL-12 with the HCV NS2 plasmid induced significantly better immune response in C57BL/6 mice. Conclusion: DNA vaccine encoding HCV NS2 is an effective candidate that can trigger CTL-based immune response against HCV. In addition, the results suggested that combining the DNA vaccine approach with immune stimulatory cytokines may significantly enhance antigen-specific immune responses. Key Words: Hepatitis C virus (HCV), NS2 protein, DNA vaccine, IL-12     

A Historic Review of the Role of CD4+ T-Cell Subsets in Development of the Immune Responses against Cutaneous and Visceral Leishmaniases

The heterogeneity of CD4⁺ T cells has been investigated since the late 1970s, when their Th1 and Th2 subsets were coined. Later studies on the cutaneous form of the Leishmaniasis were focused on the experimental models of Leishmania major infection using the susceptible BALB/c and the resistant C57BL/6 mice. At the early 21^st century, the T_reg subpopulation was introduced and its role in concomitant immunity, responsible for lifelong resistance of the host to the reinfection was proposed. Subsequent studies, mainly focused on the visceral form of the infection pointed to the role of IL-17, produced by Th17 subset of CD4⁺ T cells that along the neutrophils were shown to have important yet equivocal functions in protection against or exacerbation of the infection. Altogether, the current knowledge indicates that the above four subsets could orchestrate the immune, the regulatory and the inflammatory responses of the host against different forms of leishmaniases. Key Words: Immunity, Leishmaniasis, T-Lymphocytes     

Cloning,Expression and Characterization of Recombinant Exotoxin A-Flagellin Fusion Protein as a New Vaccine Candidate against Pseudomonas aeruginosa Infections

Background: Infections due to Pseudomonas aeruginosa are among the leading causes of morbidity and mortality in patients who suffer from impaired immune responses and chronic diseases such as cystic fibrosis. At present, aggressive antibiotic therapy is the only choice for management of P. aeruginosa infections, but emergence of highly resistant strains necessitated the development of novel alternative therapeutics including an effective vaccine. Several P. aeruginosa antigens have been tested for vaccine development, including lipopolysaccharide alone, polysaccharides alginate, extracellular proteins, exotoxin A (exo A) and killed whole cell. However, none of them are currently available clinically. Methods: In this research, recombinant exoA-flagellin (fliC) fusion protein as a cocktail antigen was expressed and purified and its antigenic characteristics were evaluated. Results: Expression of recombinant fusion protein by E. coli using pET22b vector resulted in production of exoA-fliC fusion protein in high concentration. Based on Western-blotting results, recombinant fusion protein showed a good antigenic interaction with sera from patients with various P. aeruginosa infections. Conclusion: These results suggested that recombinant exoA-fliC fusion protein can be produced in the laboratory, and tested as a candidate vaccine in P. aeruginosa infections. Key Words: Pseudomonas aeruginosa, Exotoxin A (exoA), Flagellin (fliC), Vaccines     

Immunosuppressive proteins isolated from spiral and coccoid cytoplasmic solutions of Helicobacter pylori

The aim of this study was to investigate the antiproliferative proteins that probably have a role in Helicobacter pylori evade of immune response and cause chronic infection disease and also to see if coccoid form had a role in its chronicity. H. pylori strain VacA s2/m2 positive and CagA negative, from a gastric biopsy of a patient with peptic ulcer disease, was isolated and cultured in brucella agar. Both spiral and coccoid forms were harvested and ruptured by sonication. The cytoplasmic solutions of both forms were collected and their fractions obtained by gel chromatography and preparative polyacrylamide gel electrophoresis. The fractions were analyzed by MTT assay for their antiproliferative activity. We isolated two proteins with a significant dose dependent antiproliferative activity that analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one of them that was urease positive showed two bands with 61 and 27 kDa, which is resumed to urease of H. pylori, another consist of 57 and 63 kDa. Helicobacterpylori secret some proteins like urease that inhibit immune cells proliferation response against its antigens.     

Distribution, fauna and seasonal variation of sandflies, simultaneous detection of nuclear internal transcribed spacer ribosomal DNA gene of Leishmania major in Rhombomys opimus and Phlebotomus papatasi, in Natanz district in central part of Iran

Background: Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major is increasing in many parts of Iran. This disease originally is a disease found in gerbils. Leishmania parasites are transmitted by sandflies that live and breed in gerbil burrows. Nested PCR amplified Leishmania ITS1-5.8S rRNA gene in both main reservoir host “Rhombomys opimus” and in the “Phlebotomus papatasi” main vector of ZCL, in Iran. Population differentiation and seasonal variation of sandflies were analyzed at a microgeographical level in order to identify any isolation by distance, habitat or seasons. Methods: Populations of sandflies were sampled from the edges of villages in Natanz, Isfahan province, Iran, using the Centers for Disease Control traps and sticky papers. Individual sandflies were identified based on external and internal morphological characters. Nested PCR protocols were used to amplify Leishmania ITS1-5.8S rRNA gene, which were shown to be species-specific via DNA sequence. Results: A total of 4500 sandflies were collected and identified. P. papatasi, Phlebotomus sergenti and Phlebotomus jacusieli from genus Phlebotomus and Sergentomyia sintoni and Sergentomyia clydei from genus Sergentomyia were identified in this region. P. papatasi was the most abundant sandfly in the collections. Ten out of 549 female P. papatasi and four out of 19 R. opimus were found to be infected with L. major. Conclusion: Seasonal activity of sandflies starts in June and ends in November. Abundance of P. papatasi was in September. Finding and molecular typing of L. major in P. papatasi and R. opimus confirmed the main vector and reservoir in this region.Key Words: Leishmania major, Sandflies, Leishmaniasis, Iran     

Transgenic Approaches to Disease Resistance in Ornamental Crops

Abstract

Viral, bacterial, and fungal diseases of ornamental plants cause major losses in productivity and quality. Chemical methods are available for control of fungal diseases, and to a lesser extent for bacterial diseases, but there are no economically effective chemical controls for viral diseases except to control vector species. Host plant resistance is an effective means of controlling plant diseases, and minimizing the necessity for the application of pesticides; however, there are many ornamentals in which no natural disease resistance is available. It is possible to introduce resistance derived from other species, or even from the pathogen itself, by genetic engineering. This allows the introduction of specific, or in some instances broad spectrum, disease resistance into plant genotypes that have been selected for desirable horticultural characters; in contrast, introduction of natural resistance by traditional breeding may take many cycles of breeding to combine disease resistance with desirable ornamental quality. This article briefly reviews existing work on transformation systems for ornamentals, and discusses the various approaches to introducing resistance to viral, bacterial, and fungal diseases, and to nematode infestations. These include pathogen-related proteins, R genes, and general pathogen resistance; anti-microbial peptides; expression of anti-pathogen antibodies; viral sequences; ribozymes; antiviral peptides; ribonucleases; and ribosome-inactivating proteins. Examples are given of application of these approaches to disease resistance in other types of crop and model plant systems, and actual or potential application to disease resistance in ornamentals. Future prospects for obtaining plants with multiple pest and disease resistances are discussed.     

Molecular cloning, expression and enzymatic assay of pteridine reductase 1 from Iranian lizard Leishmania

Background: Currently, there are no effective vaccines against leishmaniasis, and treatment using pentavalent antimonial drugs is occasionally effective and often toxic for patients. The PTR1 enzyme, which causes antifolate drug resistance in Leishmania parasites encoded by gene pteridine reductase 1 (ptr1). Since Leishmania lacks pteridine and folate metabolism, it cannot synthesize the pteridine moiety from guanine triphosphate. Therefore, it must produce pteridine using PTR1, an essential part of the salvage pathway that reduces oxidized pteridines. Thus, PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy. The aim of this study was the cloning, expression, and enzymatic assay of the ptr1 gene from Iranian lizard Leishmania as a model for further studies on Leishmania. Methods: Promastigote DNA was extracted from the Iranian lizard Leishmania, and the ptr1 gene was amplified using specific primers. The PCR product was cloned, transformed into Escherichia coli strain JM109, and expressed. The recombinant protein (PTR1 enzyme) was then purified and assayed. Results: ptr1 gene was successfully amplified and cloned into expression vector. Recombinant protein (PTR1 enzyme) was purified using affinity chromatography and confirmed by Western-blot and dot blot using anti-Leishmania major PTR1 antibody and anti-T7 tag monoclonal antibody, respectively. The enzymatic assay was confirmed as PTR1 witch performed using 6-biopterin as a substrate and nicotinamide adenine dinucleotide phosphate as a coenzyme. Conclusion: Iranian lizard Leishmania ptr1 was expressed and enzymatic assay was performed successfully. Key Words: Pteridine reductase 1 (PTR1), Leishmania, Gene expression     

Antiparasitic bromotyrosine derivatives from the marine sponge Verongula rigida

Nine bromotyrosine-derived compounds were isolated from the Caribbean marine sponge Verongula rigida. Two of them, aeroplysinin-1 (1) and dihydroxyaerothionin (2), are known compounds for this species, and the other seven are unknown compounds for this species, namely: 3,5-dibromo-N,N,N-trimethyltyraminium (3), 3,5-dibromo-N,N,N, O-tetramethyltyraminium (4), purealidin R (5), 19-deoxyfistularin 3 (6), purealidin B (7), 11-hydroxyaerothionin (8) and fistularin-3 (9). Structural determination of the isolated compounds was performed using one- and two-dimensional NMR, MS and other spectroscopy data. All isolated compounds were screened for their in vitro activity against three parasitic protozoa: Leishmania panamensis, Plasmodium falciparum and Trypanosoma cruzi. Compounds 7 and 8 showed selective antiparasitic activity at 10 and 5 μM against Leishmania and Plasmodium parasites, respectively. Cytotoxicity of these compounds on a human promonocytic cell line was also assessed.     

Immunogenicity of a Fusion Protein Comprising Coli Surface Antigen 3 and Labile B Subunit of Enterotoxigenic Escherichia coli

Background: Enterotoxigenic Escherichia coli (ETEC) strains are the major causes of diarrheal disease in humans and animals. Colonization factors and enterotoxins are the major virulence factors in ETEC pathogenesis. For the broad-spectrum protection against ETEC, one could focus on colonization factors and non-toxic heat labile as a vaccine candidate. Methods: A fusion protein is composed of a major fimbrial subunit of coli surface antigen 3, and the heat-labile B subunit (LTB) was constructed as a chimeric immunogen. For optimum level expression of protein, the gene was synthesized with codon bias of E. coli. Also, recombinant protein was expressed in E. coli BL21DE3. ELISA and Western tests were carried out for determination of antigen and specificity of antibody raised against recombinant protein in animals. The anti-toxicity and anti-adherence properties of the immune sera against ETEC were also evaluated. Results: Immunological analyses showed the production of high titer of specific antibody in immunized mice. The built-in LTB retains native toxin properties which were approved by GM₁ binding assay. Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells. Conclusion: The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response. The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation. Key Words: Recombinant vaccine, Enterotoxigenic Escherichia coli (ETEC), cstH, eltB     

The Application of Mucoadhesive Chitosan Nanoparticles in Nasal Drug Delivery

Mucosal delivery of antigens can induce both humoral and cellular immune responses. Particularly, the nasal cavity is a strongly inductive site for mucosal immunity among several administration routes, as it is generally the first point of contact for inhaled antigens. However, the delivery of antigens to the nasal cavity has some disadvantages such as rapid clearance and disposition of inhaled materials. For these reasons, remarkable efforts have been made to develop antigen delivery systems which suit the nasal route. The use of nanoparticles as delivery vehicles enables protection of the antigen from degradation and sustains the release of the loaded antigen, eventually resulting in improved vaccine and/or drug efficacy. Chitosan, which exhibits low toxicity, biodegradability, good cost performance, and strong mucoadhesive properties, is a useful material for nanoparticles. The present review provides an overview of the mucosal immune response induced by nanoparticles, recent advances in the use of nanoparticles, and nasal delivery systems with chitosan nanoparticles.     

Transfer of efficient anti-melanocyte T cells from vitiligo donors to melanoma patients as a novel immunotherapeutical strategy

Background

Vitiligo is a relatively common progressive depigmentary condition that is believed to be due to the autoimmune-mediated loss of epidermal melanocytes. High frequencies of self-reactive T lymphocytes directed toward melanocyte differentiation antigens are found in vitiligo patients and might be directly responsible for the pathogenesis of the disease. An interesting aspect of vitiligo is its relation to melanoma: cytotoxic T lymphocytes directed to self antigens shared by normal melanocytes and melanoma cells are found in both conditions, but the resulting immune reactions are completely different. From this standpoint, the selective destruction of pigment cells that occurs in cases of vitiligo is the therapeutic goal sought in melanoma research.

Presentation of the hypothesis

Our working hypothesis is that vitiligo patients might represent a unique source of therapeutic cells to be used in allo-transfer for HLA-matched melanoma patients. The adoptive transfer of ex-vivo generated autologous tumor-specific T cells is a therapy that has met with only limited success, essentially because of inability to isolate therapeutically valuable T cells from the majority of tumor patients. Ideally, model systems where strong and efficient responses against the same (tumor) antigens are achieved would represent a better source of therapeutic cells. We believe it is possible to identify one such model in the melanoma-vitiligo dichotomy: T lymphocytes specific for different melanocyte differentiation antigens are found in vitiligo and represent the effective anti-melanocyte reactivity that is often ineffective in melanoma.

Testing the hypothesis

Melanocyte-specific T cell clones can be isolated from the peripheral blood of vitiligo patients and tested for their capacity to efficiently expand in vitro without loosing their cytotoxic activity and to migrate to the skin. Cytotoxicity against melanoma patients' non-tumor cells can also be tested. In addition, it would be interesting to attempt an in vivo animal model. If the results obtained from these validation steps will be satisfactory, it might be possible to plan the clinical grade preparation of relevant clones for transfer.

Implications of the hypothesis

When translated into a clinical trial, the possibility of in vitro selecting few effective tumor-specific T cell clones for infusion, inherent with this approach, could enhance the therapeutic graft-versus-tumor effect while possibly decreasing the risk of graft-versus-host disease.

     

Gluten and non-gluten proteins of wheat as target antigens in autism,Crohn’s and celiac disease

Studies show that patients with celiac disease react not only with gluten wheat proteins but also with non-gluten wheat components. Our goal was to measure IgG or IgA antibodies against wheat proteins or peptides that would provide the most sensitive method for the detection of wheat immune reaction in children with autism spectrum disorder, and patients with Crohn’s and celiac disease (CD). Using ELISA, we measured these antibodies against various gluten and non-gluten wheat proteins. Compared to controls in all three conditions, the strongest reaction was against CXCR3-binding gliadin peptide, followed by a wheat protein mixture, and α-gliadin 33-mer peptide. We determined that a sample that strongly reacted against non-gluten proteins also reacted strongly against gluten proteins. We also found that IgA antibodies against CXCR3-binding gliadin peptide were strongly reactive in a subgroup of 33% in the autism group, 42% in the Crohn’s group, and all tested samples with CD. The results indicate that measuring IgG and IgA antibodies against non-gluten proteins adds nothing to the pathologic relevance of these antibodies. Further research is needed on CXCR3-binding gliadin peptide antibodies as a possible biomarker and as a guide for dietary elimination in CD, Crohn’s disease and a subgroup of children with ASD.     

Effect of dietary garlic on immune response of broiler chicks to live Newcastle Disease vaccine

This study was designed to determine the effect of garlic powder on humoral immune response of broilers against NDV (Newcastle Disease Virus) vaccine. Two hundred and forty, two-day-old, Ross chicks were randomly assigned into 4 groups of 60 birds each. Chicks in groups 1 and 2 were given control mash diet during the experiment (6 week), but those in groups 3 and 4 were fed on control diet supplemented with 1 and 3% garlic powder, respectively. All groups except number 1 were vaccinated by eye-drop with B1 strain (Pestikal, Croatia) at 9 and 18 days of age. Ten blood samples were taken from each group on days 0, 14, 24 and 34 after first vaccination. The serum antibody level against NDV was measured by both HI and ELISA tests. The EDTA-mixed blood samples were examined for total and differential leukocyte count. The results showed that antibody titers in vaccinated chicks were significantly more than in non-vaccinated chicks (p < 0.05), but not influenced by the diet (p > 0.05). A significant increase of total leukocyte and percentage of lymphocytes was observed in vaccinated chicks 14 days after vaccination, but there were no difference (p > 0.05) among vaccinated groups. It is concluded that inclusion of garlic powder to the diet of broilers don't have any beneficial effect on humoral immune response to live NDV vaccine.     

Expression of the herpes simplex virus type 2 glycoprotein D in baculovirus expression system and evaluation of its immunogenicity in guinea pigs

Background: Herpes simplex virus type 2 (HSV-2) is highly prevalent and major cause of genital herpes in humans. The life-long nature of infection and the increasing prevalence of genital herpes imply that vaccination is the best strategy for controlling the spread of infection and limiting HSV disease. HSV glycoprotein D (gD) is one of the most important viral immunogen which has an essential role in virus infectivity and induction of immune responses. Methods: HSV-2 DNA was extracted and used as template in polymerase chain reactions to amplify gD2 gene. The PCR product was confirmed by restriction enzyme analysis, cloned into a cloning vector and then sequenced. The Bac-to-Bac expression system was used to express HSV-2 gD in insect cells. The expressed protein was used as subunit vaccine to immunize guinea pigs after confirmation. Results: The expressed protein was confirmed with SDS-PAGE and Western-blot analysis. In Western-blot analysis, two major protein bands, with approximate molecular weights of 52-55 and 41-43 kDa corresponding to the glycosylated and non-glycosylated forms of gD2 protein, were observed, respectively. Immunization with the recombinant gD2 could elicit humoral responses in guinea pigs as measured by neutralization test and ELISA, and offered high protection against induced HSV-2 genital disease. Conclusion: The baculovirus expression of heterologous genes permits proper folding, post-translational modification and oligomerization in manners that are often identical to those that occur in mammalian cells. Expression of proteins under the control of the strong polyhedrin promoter, allowing high level protein production, can be used as subunit vaccine.     

Leishmania Parasite: the Impact of New Serum-Free Medium as an Alternative for Fetal Bovine Serum

Background:Flagellated protozoan of the genus Leishmania is the causative agent of vector-borne parasitic diseases of leishmaniasis. Since the production of recombinant pharmaceutical proteins requires the cultivation of host cells in a serum-free medium, the elimination of FBS can improve the possibility of large-scale culture of Leishmania parasite. In the current study, we aimed at evaluating a new serum-free medium in Leishmania parasite culture for future live Leishmania vaccine purposes. Methods:Recombinant L. tarentolae secreting PpSP15-EGFP and wild type L. major were cultured in serum-free (CSFM) and serum-supplemented medium. The growth rate, protein expression, and infectivity of cultured parasites in both conditions was then evaluated and compared. Results:Diff-Quick staining and epi-fluorescence microscopy examination displayed the typical morphology of L. major and L. tarentolae-PpSP15-EGFP promastigote grown in CSFM medium. The amount of EGFP expression was similar in CSMF medium compared to M199 supplemented with 5% FBS in flow cytometry analysis of L. tarentolae-PpSP15-EGFP parasite. Also, a similar profile of PpSP15-EGFP proteins was recognized in Western blot analysis of L. tarentolae-PpSP15-EGFP cultured in CSMF and the serum-supplemented medium. Footpad swelling and parasite load measurements showed the ability of CSFM medium to support the L. major infectivity in BALB/C mice. Conclusion:This study demonstrated that CSFM can be a promising substitute for FBS supplemented medium in parasite culture for live vaccination purposes. Key Words: Growth rate, L. major, L. tarentolae, Serum-free medium, PpSP15-EGFP protein     

Metabolomics-Based Study of Logarithmic and Stationary Phases of Promastigotes in Leishmania major by 1H NMR Spectroscopy

Background:

Cutaneous leishmaniasis is one of the most important parasitic diseases in humans. In this disease, one of the responsible organisms is Leishmania major, which is transmitted by sandfly vector. There are specific differences in biochemical profiles and metabolite pathways in logarithmic and stationary phases of Leishmania parasites. In the present study, ¹H NMR spectroscopy was used to examine the metabolites outliers in the logarithmic and stationary phases of promastigotes in L. major to enlighten more about the transmission mechanism in metacyclogenesis of L. major.

Methods:

Promastigote was cultured, logarithmic and stationary phases were separated by the peanut agglutinin, and cell metabolites were extracted. ¹H NMR spectroscopy was applied, and outliers were analyzed using principal component analysis.

Results:

The most altered metabolites in stationary and logarithmic phases were limited to citraconic acid, isopropylmalic acid, L-leucine, ornithine, caprylic acid, capric acid, and acetic acid.

Conclusion:

¹H NMR spectroscopy could play an important role in the characterization of metabolites in biochemical pathways during a metacyclogenesis process. These metabolites and their pathways can help in exploiting a transmission mechanism in metacyclogenesis, and outcoming data might be used in the metabolic network reconstruction of L. major modeling. Key Words: Leishmania major, Metabolomics, Principal component analysis     

Horizontal resistance in tomato against the Meloidogyne-Fusarium complex: an artefact of parasitic epistasis

In a disease complex, epistatic and hypostatic parasites interact and modify the host response in such a way that the final disease reaction mimics quantitative reaction types. Data from an experiment using Meloidogyne incognita (Kofoid and White) Chitwood and Fusarium oxysporum f. lycopersici Snyd. and Hans. on tomato support the hypothesis that horizontal resistance against root pathogens may be an artefact of disease complexes. The implications, for crop protection, of the occurrence of disease complexes are discussed.     

Broomrape management in faba bean

Broomrapes (Orobanche spp. and Phelipanche spp.) are weedy root parasites that represent a major constraint for faba bean (Vicia faba) cultivation in the Mediterranean area. Control methods are being developed that comprise techniques ranging from agronomical practices to precision farming, including chemical and biological control, genetic and induced resistance, modelling, and probably nanotechnology in a nearby future. However, the main concern is that to date, no single method of control provides complete protection against these parasites. For that reason, an integrated approach is needed in which a variety of such techniques are combined, in order to maintain parasite populations below threshold levels of damage.