Sequence diversity and evolutionary dynamics of the dimorphic antigen merozoite surface protein-6 and other Msp genes of Plasmodium falciparum

Immune evasion by Plasmodium falciparum is favored by extensive allelic diversity of surface antigens. Some of them, most notably the vaccine-candidate merozoite surface protein (MSP)-1, exhibit a poorly understood pattern of allelic dimorphism, in which all observed alleles group into two highly diverged allelic families with few or no inter-family recombinants. Here we describe contrasting levels and patterns of sequence diversity in genes encoding three MSP-1-associated surface antigens of P. falciparum, ranging from an ancient allelic dimorphism in the Msp-6 gene to a near lack of allelic divergence in Msp-9 to a more classical multi-allele polymorphism in Msp-7 Other members of the Msp-7 gene family exhibit very little polymorphism in non-repetitive regions. A comparison of P. falciparum Msp-6 sequences to an orthologous sequence from P. reichenowi provided evidence for distinct evolutionary histories of the 5` and 3` segments of the dimorphic region in PfMsp-6, consistent with one dimorphic lineage having arisen from recombination between now-extinct ancestral alleles. In addition. we uncovered two surprising patterns of evolution in repetitive sequence. Firsts in Msp-6, large deletions are associated with (nearly) identical sequence motifs at their borders. Second, a comparison of PfMsp-9 with the P. reichenowi ortholog indicated retention of a significant inter-unit diversity within an 18-base pair repeat within the coding region of P. falciparum, but homogenization in P. reichenowi. (C) 2009 Elsevier B.V. All rights reserved.

Fonsecaea pedrosoi infection induces differential modulation of costimulatory molecules and cytokines in monocytes from patients with severe and mild forms of chromoblastomycosis

The host defense mechanism in chromoblastomycosis has not been thoroughly investigated. It has been suggested that cell- mediated immunity in patients with long- standing chromoblastomycosis is somehow impaired. As a result, these individuals became unable to develop an efficient immune reaction. Many studies have shown that monocyte- derived macrophages exhibit critical activities in immunity to microorganisms. Moreover, the ability of cells from the monocytic lineage to process and present antigens, to produce cytokines, and to provide costimulatory signals confirms their pivotal role in the initiation of specific immune responses. In the present study, it was observed that monocytes from patients with a severe form of disease had a higher production of IL- 10 and a lower expression of HLA- DR and costimulatory molecules when stimulated with specific antigen or LPS. Immune modulation with recombinant IL- 12 or anti- IL- 10 can restore the antigen- specific Th1- type immune response in chromoblastomycosis patients by up- regulating HLA- DR and costimulatory molecules in monocytes. Therefore, our data show that monocytes from patients with different clinical forms of chromoblastomycosis present distinct phenotypic and functional profiles. This observation suggests possible mechanisms that control the T cell response and influence their role in the development of pathology.

The monoclonal antibody against the major diagnostic antigen of Paracoccidioides brasiliensis mediates immune protection in infected BALB/c mice challenged intratracheally with the fungus

The protective role of specific antibodies against Paracoccidioides brasiliensis is controversial. In the present study, we analyzed the effects of monoclonal antibodies on the major diagnostic antigen (gp43) using in vitro and in vivo P. brasiliensis infection models. The passive administration of some monoclonal antibodies (MAbs) before and after intratracheal or intravenous infections led to a reduced fungal burden and decreased pulmonary inflammation. The protection mediated by MAb 3E, the most efficient MAb in the reduction of fungal burden, was associated with the enhanced phagocytosis of P. brasiliensis yeast cells by J774.16, MH-S, or primary macrophages. The ingestion of opsonized yeast cells led to an increase in NO production by macrophages. Passive immunization with MAb 3E induced enhanced levels of gamma interferon in the lungs of infected mice. The reactivity of MAb 3E against a panel of gp43-derived peptides suggested that the sequence NHVRIPIGWAV contains the binding epitope. The present work shows that some but not all MAbs against gp43 can reduce the fungal burden and identifies a new peptide candidate for vaccine development.

The leptospiral antigen Lp49 is a two-domain protein with putative protein binding function

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 angstrom resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date. (C) 2008 Elsevier Inc. All rights reserved.

Ki-67 and Bcl-2 Antigen Expression in Adenomatous Colorectal Polyps from Women with Breast Cancer

Background. The aim of this study was to evaluate Ki-67 and Bcl-2 antigen expression in colorectal polyps from women with breast cancer. Methods. A randomized, controlled study was carried out in 35 women, either with or without breast cancer, who had adenomatous colorectal polyps. The patients were divided into two groups: group A (without breast cancer; control group; n = 17) and group B (with breast cancer; study group; n = 18). Immunohistochemistry was performed on the colorectal polyps to evaluate Ki-67 and Bcl-2 antigen expression. Student`s t-test and the chi(2) test were used for the statistical analysis of Ki-67 and Bcl-2 expression, respectively. Statistical significance was established as P < 0.05. Results. The mean percentage of Ki-67-stained nuclei in groups A and B was 36.25 +/- 2.31 and 59.44 +/- 3.34 ( SEM), respectively (P < 0.0001), while the percentage of cases with cells expressing Bcl-2 in groups A and B was 23.5 and 77.8%, respectively (P < 0.001). Conclusions. In the present study, there was greater proliferative activity and greater expression of the antiapoptotic protein Bcl-2 in the colorectal polyps of women with breast cancer.

Effects of Raloxifene on Ki-67 and CD34 Antigen Expression in Breast Cancer

Background: The aim of this study was to evaluate the effect of raloxifene on CD34 and Ki-67 antigen expression in breast cancer specimens from postmenopausal women. Methods: Sixteen postmenopausal patients with operable, stage II (>= 3 cm), estrogen receptor-positive breast cancer, who took 60 mg of raloxifene daily for 28 days, participated in this study. Immunohistochemistry was carried out in tumor samples prior to and following raloxifene treatment to evaluate CD34 and Ki-67 protein expression. Angiogenesis was quantified in 10 randomly selected fields per slide, and Ki-67-stained nuclei were counted in 1,000 cells per slide using an image capture and analysis system with 400 ! magnification. Student`s t test for paired samples was used for the statistical analysis of data. Statistical significance was established at p < 0.05. Results: The mean number of microvessels was 44.44 +/- 3.54 prior to raloxifene therapy and 22.63 +/- 1.61 following therapy (p < 0.001), and the mean percentage of Ki-67-stained nuclei was 19.28 +/- 8 1.61 and 12.13 +/- 8 1.48 prior to and following raloxifene treatment, respectively (p < 0.001). Conclusion: Raloxifene significantly reduces CD34 and Ki-67 protein expression in breast carcinoma in postmenopausal women. Copyright (C) 2008 S. Karger AG, Basel

Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp.

Leptospixosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospires, where it accounts for up to 75% of the total outer membrane proteins. It is highly immunogenic, and recent studies have implicated LipL32 as an extracellular matrix binding protein, interacting with collagens, fibronectin, and laminin. In order to better understand the biological role and the structural requirements for the function of this important lipoprotein, we have determined the 2.25-angstrom-resolution structure of recombinant LipL32 protein corresponding to residues 21-272 of the wild-type protein (LipL32(21-272)). The LipL32(21-272) monomer is made of a jelly-roll fold core from which several peripheral secondary structures protrude. LipL32(21-272) is structurally similar to several other jelly-roll proteins, some of which bind calcium ions and extracellular matrix proteins. Indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence, and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium-binding properties of LipL32(21-272). Ca(2+) binding resulted in a significant increase in the thermal stability of the protein, and binding was specific for Ca(2+) as no structural or stability perturbations were observed for Mg(2+), Zn(2+), or Cu(2+). Careful examination of the crystal lographic structure suggests the locations of putative regions that could mediate Ca(2+) binding as well as binding to other interacting host proteins, such as collagens, fibronectin, and lamixidn. (C) 2009 Elsevier Ltd. All rights reserved.

Sources of doubt, sources of duty: HLA Hart on international law

In many ways HLA Hart’s critical analysis of the concept of law, with its repudiation of simple command theories of legal obligation, is at the same time a critique of the notion of state sovereignty. It is therefore an adumbration, if no more, of a radical reconceptualisation of international law, one which redefines the distinction between municipal and in-ternational jurisdiction. This paper is an exploration of what Hart could or ‘should’ have said about international law, based as much as possible on what he did say about international law and law in general. After some introductory comments it outlines Hart’s general analysis of law, with particular reference to the relevance for our understanding of international law.

Unifying manufacturing simulation models using HLA

The increasing use of simulation in manufacturing has seen an increase in simulation models created using many simulation package. This use of different simulators can create simulation islands in a manufacturing factory, making it difficult to get a true simulated overview of the factory. At present, there are only a few cases where manufacturing simulations have been linked to enable multiple simulation models to run as one. This research expands upon these cases. For this paper the topic of discussion is the research in connecting different 'Commercial Off The Shelf' simulators together to allow flow of all information through the connected models using high level architecture.

Linking discrete event simulation models using HLA

The increasing usage of discrete event simulation packages for modeling and analyzing manufacturing and logistics has led to a need for connecting simulation models together at runtime. One such methodology for linking discrete event simulation models together has been developed for this research and this paper demonstrates the usage of this linking method. A unified simulation model is developed from two submodels developed using different simulation packages.

Evidence for a common role for the serine-type Plasmodium falciparum serine repeat antigen proteases : implications for vaccine and drug design

Serine repeat antigens (SERAs) are a family of secreted “cysteine-like” proteases of Plasmodium parasites. Several SERAs possess an atypical active-site serine residue in place of the canonical cysteine. The human malaria parasite Plasmodium falciparum possesses six “serine-type” (SERA1 to SERA5 and SERA9) and three “cysteine-type” (SERA6 to SERA8) SERAs. Here, we investigate the importance of the serine-type SERAs to blood-stage parasite development and examine the extent of functional redundancy among this group. We attempted to knock out the four P. falciparum serine-type SERA genes that have not been disrupted previously. SERA1, SERA4, and SERA9 knockout lines were generated, while only SERA5, the most strongly expressed member of the SERA family, remained refractory to genetic deletion. Interestingly, we discovered that while SERA4-null parasites completed the blood-stage cycle normally, they exhibited a twofold increase in the level of SERA5 mRNA. The inability to disrupt SERA5 and the apparent compensatory increase in SERA5 expression in response to the deletion of SERA4 provides evidence for an important blood-stage function for the serine-type SERAs and supports the notion of functional redundancy among this group. Such redundancy is consistent with our phylogenetic analysis, which reveals a monophyletic grouping of the serine-type SERAs across the genus Plasmodium and a predominance of postspeciation expansion. While SERA5 is to some extent further validated as a target for vaccine and drug development, our data suggest that the expression level of other serine-type SERAs is the only barrier to escape from anti-SERA5-specific interventions.

Whole blood analysis of phagocytosis, apoptosis, cytokine production, and leukocyte subsets in healthy older men and women: the ZENITH study

Few studies to date have examined age-related changes in markers of immune status in healthy older individuals. The immune status of 93 healthy individuals aged 55–70 years was assessed by two- and three-color flow cytometry and biochemical analysis. There were significant age effects (p ≤.05) on monocyte phagocytic activity and cluster of differentiation (CD) 3/human leukocyte antigen-D-related (HLA-DR) late-activated T lymphocytes (% expression). There was a significant (p ≤ 0.1) Age x Sex interaction in absolute counts (x 10⁹/L) of CD3/CD8 total cytotoxic T lymphocytes (CTL), the CD4 T- helper to CD8 CTL ratio, the CD3/CD4/CD45RA naïve T helper to CD3/CD4/CD45RO memory T helper lymphocyte ratio, and interleukin (IL)-1ß (% expression) by activated monocytes. The study shows that alterations in markers of immune status occur between 55 and 70 years, and provides reference values for the lymphocyte measures in healthy men and postmenopausal women in this age group. The study further highlights the need for sex-specific reference ranges for such markers.

A new rodent model to assess blood stage immunity to the plasmodium falciparum antigen merozoite surface protein 119 reveals a protective role for invasion inhibitory antibodies

Antibodies capable of inhibiting the invasion of Plasmodium merozoites into erythrocytes are present in individuals that are clinically immune to the malaria parasite. Those targeting the 19-kD COOH-terminal domain of the major merozoite surface protein (MSP)-119 are a major component of this inhibitory activity. However, it has been difficult to assess the overall relevance of such antibodies to antiparasite immunity. Here we use an allelic replacement approach to generate a rodent malaria parasite (Plasmodium berghei) that expresses a human malaria (Plasmodium falciparum) form of MSP-119. We show that mice made semi-immune to this parasite line generate high levels of merozoite inhibitory antibodies that are specific for P. falciparum MSP-119. Importantly, protection from homologous blood stage challenge in these mice correlated with levels of P. falciparum MSP-119–specific inhibitory antibodies, but not with titres of total MSP-119–specific immunoglobulins. We conclude that merozoite inhibitory antibodies generated in response to infection can play a significant role in suppressing parasitemia in vivo. This study provides a strong impetus for the development of blood stage vaccines designed to generate invasion inhibitory antibodies and offers a new animal model to trial P. falciparum MSP-119 vaccines.