Successful Amelioration of Mitochondrial Optic Neuropathy Using the Yeast NDI1 Gene in a Rat Animal Model

Background

Leber''s hereditary optic neuropathy (LHON) is a maternally inherited disorder with point mutations in mitochondrial DNA which result in loss of vision in young adults. The majority of mutations reported to date are within the genes encoding the subunits of the mitochondrial NADH-quinone oxidoreductase, complex I. Establishment of animal models of LHON should help elucidate mechanism of the disease and could be utilized for possible development of therapeutic strategies.

Methodology/Principal Findings

We established a rat model which involves injection of rotenone-loaded microspheres into the optic layer of the rat superior colliculus. The animals exhibited the most common features of LHON. Visual loss was observed within 2 weeks of rotenone administration with no apparent effect on retinal ganglion cells. Death of retinal ganglion cells occurred at a later stage. Using our rat model, we investigated the effect of the yeast alternative NADH dehydrogenase, Ndi1. We were able to achieve efficient expression of the Ndi1 protein in the mitochondria of all regions of retinal ganglion cells and axons by delivering the NDI1 gene into the optical layer of the superior colliculus. Remarkably, even after the vision of the rats was severely impaired, treatment of the animals with the NDI1 gene led to a complete restoration of the vision to the normal level. Control groups that received either empty vector or the GFP gene had no effects.

Conclusions/Significance

The present study reports successful manifestation of LHON-like symptoms in rats and demonstrates the potential of the NDI1 gene therapy on mitochondrial optic neuropathies. Our results indicate a window of opportunity for the gene therapy to be applied successfully after the onset of the disease symptoms.     

Bone Marrow-Derived Cells May Not Be the Original Cells for Carcinogen-Induced Mouse Gastrointestinal Carcinomas

Aim

It has been reported that bone marrow-derived cells (BMDC) can be original cells of mouse gastric cancers induced by Helicobacter felis (H. felis) infection. However, it is unknown whether BMDCs are also the original cells of mouse gastrointestinal cancers induced by gastric carcinogens N-nitroso-N-methylurea (NMU) and H. felis infection.

Methods

C57BL/6 recipient mice were initially irradiated with 10Gy X-ray, reconstituted with bone marrow cells from the C57BL/6-Tg (CAG-EGFP) donor mice to label BMDCs with green fluorescence protein (GFP). After 4 weeks of recovery, the bone marrow-transplanted mice were given NMU in drinking water (240 ppm) and subsequently infected with H. felis by gavage. Eighty weeks later, all mice were euthanized for pathological examination. The BMDCs expressing GFP were detected in tissues using direct GFP fluorescence confocal microscopy analysis and immunohistochemistry staining (IHC) assays.

Results

Neoplastic lesions were induced by NMU treatment and/or H. felis infection at the antrum of the glandular stomach and small intestine. In the direct GFP fluorescence confocal assay, GFP(+) epithelial cell cluster or glands were not observed in these gastrointestinal tumors, however, most GFP(+) BMDCs sporadically located in the tumor stromal tissues. Some of these GFP(+) stromal BMDCs co-expressed the hematopoietic marker CD45 or myofibroblasts markers αSMA and SRF. In the indirect GFP IHC assay, similar results were observed among 11 gastric intraepithelial neoplasia lesions and 2 small intestine tumors.

Conclusion

These results demonstrated that BMDCs might not be the source of gastrointestinal tumor cells induced by NMU and/or H. felis infection.     

RNAi-mediated CD40-CD154 interruption promotes tolerance in autoimmune arthritis

Introduction

We have previously demonstrated that ex vivo inhibition of costimulatory molecules on antigen-pulsed dendritic cells (DCs) can be useful for induction of antigen-specific immune deviation and suppression of autoimmune arthritis in the collagen induced arthritis (CIA) model. The current study evaluated a practical method of immune modulation through temporary systemic inhibition of the costimulatory molecule CD40.

Methods

Mice with collagen II (CII)-induced arthritis (CIA) were administered siRNA targeting the CD40 molecule. Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses.

Results

Systemic administration of CD40-targeting siRNA can inhibit antigen-specific T cell response to collagen II, as well as prevent pathogenesis of disease in both a pre- and post-immunization manner in the CIA model. Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells.

Conclusions

These studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity.     

A Decreased Frequency of Regulatory T Cells in Patients with Common Variable Immunodeficiency

Introduction

Common variable immunodeficiency disorder (CVID) is a heterogeneous syndrome, characterized by deficient antibody production and recurrent bacterial infections in addition abnormalities in T cells. CD4⁺CD25^high regulatory T cells (Treg) are essential modulators of immune responses, including down-modulation of immune response to pathogens, allergens, cancer cells and self-antigens.

Objective

In this study we set out to investigate the frequency of Treg cells in CVID patients and correlate with their immune activation status.

Materials and Methods

Sixteen patients (6 males and 10 females) with CVID who had been treated with regular intravenous immunoglobulin and 14 controls were enrolled. Quantitative analyses of peripheral blood mononuclear cells (PBMC) were performed by multiparametric flow cytometry using the following cell markers: CD38, HLA-DR, CCR5 (immune activation); CD4, CD25, FOXP3, CD127, and OX40 (Treg cells); Ki-67 and IFN-γ (intracellular cytokine).

Results

A significantly lower proportion of CD4⁺CD25^highFOXP3 T cells was observed in CVID patients compared with healthy controls (P<0.05). In addition to a higher proportion of CD8⁺ T cells from CVID patients expressing the activation markers, CD38⁺ and HLA-DR⁺ (P<0.05), we observed no significant correlation between Tregs and immune activation.

Conclusion

Our results demonstrate that a reduction in Treg cells could have impaired immune function in CVID patients.     

Colonic Immune Stimulation by Targeted Oral Vaccine

Background

Currently, sufficient data exist to support the use of lactobacilli as candidates for the development of new oral targeted vaccines. To this end, we have previously shown that Lactobacillus gasseri expressing the protective antigen (PA) component of anthrax toxin genetically fused to a dendritic cell (DC)-binding peptide (DCpep) induced efficacious humoral and T cell-mediated immune responses against Bacillus anthracis Sterne challenge.

Methodology/Principal Finding

In the present study, we investigated the effects of a dose dependent treatment of mice with L. gasseri expressing the PA-DCpep fusion protein on intestinal and systemic immune responses and confirmed its safety. Treatment of mice with different doses of L. gasseri expressing PA-DCpep stimulated colonic immune responses, resulting in the activation of innate immune cells, including dendritic cells, which induced robust Th1, Th17, CD4⁺Foxp3⁺ and CD8⁺Foxp3⁺ T cell immune responses. Notably, high doses of L. gasseri expressing PA-DCpep (10¹² CFU) were not toxic to the mice. Treatment of mice with L. gasseri expressing PA-DCpep triggered phenotypic maturation and the release of proinflammatory cytokines by dendritic cells and macrophages. Moreover, treatment of mice with L. gasseri expressing PA-DCpep enhanced antibody immune responses, including IgA, IgG₁, IgG_2b, IgG_2c and IgG₃. L. gasseri expressing PA-DCpep also increased the gene expression of numerous pattern recognition receptors, including Toll-like receptors, C-type lectin receptors and NOD-like receptors.

Conclusion/Significance

These findings suggest that L. gasseri expressing PA-DCpep has substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration and may be used as a safe oral vaccine against anthrax challenge.     

CD8+ T cells and IFN-γ mediate the time-dependent accumulation of infected red blood cells in deep organs during experimental cerebral malaria

Background

Infection with Plasmodium berghei ANKA (PbA) in susceptible mice induces a syndrome called experimental cerebral malaria (ECM) with severe pathologies occurring in various mouse organs. Immune mediators such as T cells or cytokines have been implicated in the pathogenesis of ECM. Red blood cells infected with PbA parasites have been shown to accumulate in the brain and other tissues during infection. This accumulation is thought to be involved in PbA–induced pathologies, which mechanisms are poorly understood.

Methods and Findings

Using transgenic PbA parasites expressing the luciferase protein, we have assessed by real-time in vivo imaging the dynamic and temporal contribution of different immune factors in infected red blood cell (IRBC) accumulation and distribution in different organs during PbA infection. Using deficient mice or depleting antibodies, we observed that CD8⁺ T cells and IFN-γ drive the rapid increase in total parasite biomass and accumulation of IRBC in the brain and in different organs 6–12 days post-infection, at a time when mice develop ECM. Other cells types like CD4⁺ T cells, monocytes or neutrophils or cytokines such as IL-12 and TNF-α did not influence the early increase of total parasite biomass and IRBC accumulation in different organs.

Conclusions

CD8⁺ T cells and IFN-γ are the major immune mediators controlling the time-dependent accumulation of P. berghei-infected red blood cells in tissues.     

Several Important In Vitro Improvements in the Amplification,Differentiation and Tracing of Fetal Liver Stem/Progenitor Cells

Objective

We previously isolated fetal liver stem/progenitor cells (FLSPCs), but there is an urgent need to properly amplify FLSPCs, effectively induce FLSPCs differentiation, and steadily trace FLSPCs for in vivo therapeutic investigation.

Methods

FLSPCs were maintained in vitro as adherent culture or soft agar culture for large-scale amplification. To direct the differentiation of FLSPCs into hepatocytes, FLSPCs were randomly divided into four groups: control, 1% DMSO-treated, 20 ng/ml HGF-treated and 1% DMSO+20 ng/ml HGF-treated. To trace FLSPCs, the GFP gene was introduced into FLSPCs by liposome-mediated transfection.

Results

For amplifying FLSPCs, the soft agar culture were more suitable than the adherent culture, because the soft agar culture obtained more homogeneous cells. These cells were with high nuclear:cytoplasmic ratio, few cell organelles, high expression of CD90.1 and CD49f, and strong alkaline phosphatase staining. For inducing FLSPCs differentiation, treatment with HGF+DMSO was most effective (P<0.05), which was strongly supported by the typical morphological change and the significant decrease of OV-6 positive cells (P<0.01). In addition, the time of indocyanine green elimination, the percentage of glycogen synthetic cells, and the expressions of ALB, G-6-P, CK-8, CK-18 and CYP450-3A1 in HGF+DMSO-treated group were higher than in any other group. For tracing FLSPCs, after the selection of stable FLSPC transfectants, GFP expression continued over successive generations.

Conclusions

FLSPCs can properly self-renew in soft agar culture and effectively differentiate into hepatocyte-like cells by HGF+DMSO induction, and they can be reliably traced by GFP expression.     

A New Adenovirus Based Vaccine Vector Expressing an Eimeria tenella Derived TLR Agonist Improves Cellular Immune Responses to an Antigenic Target

Background

Adenoviral based vectors remain promising vaccine platforms for use against numerous pathogens, including HIV. Recent vaccine trials utilizing Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections in vaccinees. This illustrates the need to develop vaccine formulations capable of generating more potent T-cell responses to HIV antigens, such as HIV-Gag, since robust immune responses to this antigen correlate with improved outcomes in long-term non-progressor HIV infected individuals.

Methodology/Principal Findings

In this study we designed a novel vaccine strategy utilizing an Ad-based vector expressing a potent TLR agonist derived from Eimeria tenella as an adjuvant to improve immune responses from a [E1-]Ad-based HIV-Gag vaccine. Our results confirm that expression of rEA elicits significantly increased TLR mediated innate immune responses as measured by the influx of plasma cytokines and chemokines, and activation of innate immune responding cells. Furthermore, our data show that the quantity and quality of HIV-Gag specific CD8⁺ and CD8⁻ T-cell responses were significantly improved when coupled with rEA expression. These responses also correlated with a significantly increased number of HIV-Gag derived epitopes being recognized by host T cells. Finally, functional assays confirmed that rEA expression significantly improved antigen specific CTL responses, in vivo. Moreover, we show that these improved responses were dependent upon improved TLR pathway interactions.

Conclusion/Significance

The data presented in this study illustrate the potential utility of Ad-based vectors expressing TLR agonists to improve clinical outcomes dependent upon induction of robust, antigen specific immune responses.     

A Flow Cytometry-Based Quantitative Drug Sensitivity Assay for All Plasmodium falciparum Gametocyte Stages

Background

Malaria elimination/eradication campaigns emphasize interruption of parasite transmission as a priority strategy. Screening for new drugs and vaccines against gametocytes is therefore urgently needed. However, current methods for sexual stage drug assays, usually performed by counting or via fluorescent markers are either laborious or restricted to a certain stage. Here we describe the use of a transgenic parasite line for assaying drug sensitivity in all gametocyte stages.

Methods

A transgenic parasite line expressing green fluorescence protein (GFP) under the control of the gametocyte-specific gene α-tubulin II promoter was generated. This parasite line expresses GFP in all gametocyte stages. Using this transgenic line, we developed a flow cytometry-based assay to determine drug sensitivity of all gametocyte stages, and tested the gametocytocidal activities of four antimalarial drugs.

Findings

This assay proved to be suitable for determining drug sensitivity of all sexual stages and can be automated. A Z’ factor of 0.79±0.02 indicated that this assay could be further optimized for high-throughput screening. The daily sensitivity of gametocytes to three antimalarial drugs (chloroquine, dihydroartemisinin and pyronaridine) showed a drastic decrease from stage III on, whereas it remained relatively steady for primaquine.

Conclusions

A drug assay was developed to use a single transgenic parasite line for determining drug susceptibility of all gametocyte stages. This assay may be further automated into a high-throughput platform for screening compound libraries against P. falciparum gametocytes.     

Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

Background

Tumor necrosis factor (TNF) is an important cytokine in the pathogenesis of inflammatory bowel disease (IBD). Anti-TNF antibodies have been successfully implemented in IBD therapy, however their efficacies differ among IBD patients. Here we investigate the influence of CD64 Fc receptor on the inhibitory activity of anti-TNFs in cells of intestinal wall.

Methods

Intestinal cell lines, monocytes/macrophages and peripheral blood mononuclear cells (PBMCs) were used as models. The efficacies of adalimumab, infliximab and certolizumab-pegol were assessed by RT-PCR for target genes. Protein levels and localizations were examined by Western blotting and immunofluorescence. Antibody fragments were obtained by proteolytic digestion, immunoprecipitation and protein chip analysis. Knock-down of specific gene expression was performed using siRNAs.

Results

Infliximab had limited efficacy towards soluble TNF in cell types expressing Fc gamma receptor CD64. Both adalimumab and infliximab had lower efficacies in PBMCs of IBD patients, which express elevated levels of CD64. Infliximab-TNF complexes were more potent in activating CD64 in THP-1 cells than adalimumab, which was accompanied by distinct phospho-tyrosine signals. Blocking Fc parts and isolation of Fab fragments of infliximab improved its efficacy. IFN-γ-induced expression of CD64 correlated with a loss of efficacy of infliximab, whereas reduction of CD64 expression by either siRNA or PMA treatment improved inhibitory activity of this drug. Colonic mRNA expression levels of CD64 and other Fc gamma receptors were significantly increased in the inflamed tissues of infliximab non-responders.

Conclusions

CD64 modulates the efficacy of infliximab both in vitro and ex vivo, whereas the presence of this receptor has no impact on the inhibitory activity of certolizumab-pegol, which lacks Fc fragment. These data could be helpful in both predicting and evaluating the outcome of anti-TNF therapy in IBD patients with elevated systemic and local levels of Fc receptors.     

CD1d-expressing breast cancer cells modulate NKT cell-mediated antitumor immunity in a murine model of breast cancer metastasis

Background

Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.

Methodology/Principal Findings

In this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors.

Conclusions/Significance

The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.     

The interaction between regulatory T cells and NKT cells in the liver: a CD1d bridge links innate and adaptive immunity

Background/Aims

Regulatory T cells (Tregs) and natural killer T (NKT) cells are two distinct lymphocyte subsets that independently regulate hepatic adaptive and innate immunity, respectively. In the current study, we examine the interaction between Tregs and NKT cells to understand the mechanisms of cross immune regulation by these cells.

Methods

The frequency and function of Tregs were evaluated in wild type and NKT cell deficient (CD1dko) mice. In vitro lymphocyte proliferation and apoptosis assays were performed with NKT cells co-cultured with Tregs. The ability of Tregs to inhibit NKT cells in vivo was examined by adoptive transfer of Tregs in a model of NKT cell mediated hepatitis.

Results

CD1dko mice have a significant reduction in hepatic Tregs. Although, the Tregs from CD1dko mice remain functional and can suppress conventional T cells, their ability to suppress activation induced NKT cell proliferation and to promote NKT cell apoptosis is greatly diminished. These effects are CD1d dependent and require cell to cell contact. Adoptive transfer of Tregs inhibits NKT cell-mediated liver injury.

Conclusions

NKT cells promote Tregs, and Tregs inhibit NKT cells in a CD1d dependent manner requiring cell to cell contact. These cross-talk immune regulations provide a linkage between innate and adaptive immunity.     

Immunomodulatory strategies prevent the development of autoimmune emphysema

Background

The presence of anti-endothelial cell antibodies and pathogenic T cells may reflect an autoimmune component in the pathogenesis of emphysema. Whether immune modulatory strategies can protect against the development of emphysema is not known.

Methods

Sprague Dawley rats were immunized with human umbilical vein endothelial cells (HUVEC) to induce autoimmune emphysema and treated with intrathymic HUVEC-injection and pristane. Measurements of alveolar airspace enlargement, cytokine levels, immuno histochemical, western blot analysis, and T cell repertoire of the lung tissue were performed.

Results

The immunomodulatory strategies protected lungs against cell death as demonstrated by reduced numbers of TUNEL and active caspase-3 positive cells and reduced levels of active caspase-3, when compared with lungs from HUVEC-immunized rats. Immunomodulatory strategies also suppressed anti-endothelial antibody production and preserved CNTF, IL-1alpha and VEGF levels. The immune deviation effects of the intrathymic HUVEC-injection were associated with an expansion of CD4+CD25+Foxp3+ regulatory T cells. Pristane treatment decreased the proportion of T cells expressing receptor beta-chain, Vβ16.1 in the lung tissue.

Conclusions

Our data demonstrate that interventions classically employed to induce central T cell tolerance (thymic inoculation of antigen) or to activate innate immune responses (pristane treatment) can prevent the development of autoimmune emphysema.     

Regulatory Phenotype,PD-1 and TLR3 Expression in T Cells and Monocytes from HCV Patients Undergoing Antiviral Therapy: A Randomized Clinical Trial

Background & Aims

The cellular immunity has a profound impact on the status of hepatitis C virus (HCV) infection. However, the response of cellular immunity on the virological response in patients with antiviral treatment remains largely unclear. We aimed to clarify the response of peripheral T cells and monocytes in chronic hepatitis C patients with antiviral treatment.

Methods

Patients with chronic hepatitis C were treated either with interferon alpha-2b plus ribavirin (n = 37) or with pegylated interferon alpha-2a plus ribavirin (n = 33) for up to 24 weeks. Frequencies of peripheral regulatory T-cells (Tregs), programmed death-1 (PD-1) expressing CD4⁺ T-cells or CD8⁺ T-cells and toll-like receptor (TLR) 3 expressing CD14⁺ monocytes were evaluated by flow cytometry in patients at baseline, 12 and 24 weeks following treatment and in 20 healthy controls.

Results

Frequencies of Tregs, PD-1 and TLR3 expressing cells were higher in patients than those in control subjects (P<0.05). Patients with complete early virological response (cEVR) showed lower Tregs, PD-1 expressing CD4⁺ or CD8⁺ T-cells than those without cEVR at 12 weeks (P<0.05). Patients with low TLR3 expressing CD14⁺ monocytes at baseline had a high rate of cEVR (P<0.05).

Conclusions

Low peripheral TLR3 expressing CD14⁺ monocytes at baseline could serve as a predictor for cEVR of antiviral therapy in chronic HCV-infected patients. The cEVR rates were significantly increased in the patients with reduced circulating Tregs, PD-1 expressing CD4⁺ or CD8⁺ T-cells.

Trial Registration

Chinese Clinical Trial Registry ChiCTR10001090.     

The Immunological and Virological Consequences of Planned Treatment Interruptions in Children with HIV Infection

Objectives

To evaluate the immunological and viral consequences of planned treatment interruptions (PTI) in children with HIV.

Design

This was an immunological and virological sub-study of the Paediatric European Network for Treatment of AIDS (PENTA) 11 trial, which compared CD4-guided PTI of antiretroviral therapy (ART) with continuous therapy (CT) in children.

Methods

HIV-1 RNA and lymphocyte subsets, including CD4 and CD8 cells, were quantified on fresh samples collected during the study; CD45RA, CD45RO and CD31 subpopulations were evaluated in some centres. For 36 (18 PTI, 18 CT) children, immunophenotyping was performed and cell-associated HIV-1 DNA analysed on stored samples to 48 weeks.

Results

In the PTI group, CD4 cell count fell rapidly in the first 12 weeks off ART, with decreases in both naïve and memory cells. However, the proportion of CD4 cells expressing CD45RA and CD45RO remained constant in both groups. The increase in CD8 cells in the first 12 weeks off ART in the PTI group was predominantly due to increases in RO-expressing cells. PTI was associated with a rapid and sustained increase in CD4 cells expressing Ki67 and HLA-DR, and increased levels of HIV-1 DNA.

Conclusions

PTI in children is associated with rapid changes in CD4 and CD8 cells, likely due to increased cell turnover and immune activation. However, children off treatment may be able to maintain stable levels of naïve CD4 cells, at least in proportion to the memory cell pool, which may in part explain the observed excellent CD4 cell recovery with re-introduction of ART.     

Beta cells within single human islets originate from multiple progenitors

Background

In both humans and rodents, glucose homeostasis is controlled by micro-organs called islets of Langerhans composed of beta cells, associated with other endocrine cell types. Most of our understanding of islet cell differentiation and morphogenesis is derived from rodent developmental studies. However, little is known about human islet formation. The lack of adequate experimental models has restricted the study of human pancreatic development to the histological analysis of different stages of pancreatic development. Our objective was to develop a new experimental model to (i) transfer genes into developing human pancreatic cells and (ii) validate gene transfer by defining the clonality of developing human islets.

Methods and Findings

In this study, a unique model was developed combining ex vivo organogenesis from human fetal pancreatic tissue and cell type-specific lentivirus-mediated gene transfer. Human pancreatic progenitors were transduced with lentiviruses expressing GFP under the control of an insulin promoter and grafted to severe combined immunodeficient mice, allowing human beta cell differentiation and islet morphogenesis. By performing gene transfer at low multiplicity of infection, we created a chimeric graft with a subpopulation of human beta cells expressing GFP and found both GFP-positive and GFP-negative beta cells within single islets.

Conclusion

The detection of both labeled and unlabeled beta cells in single islets demonstrates that beta cells present in a human islet are derived from multiple progenitors thus providing the first dynamic analysis of human islet formation during development. This human transgenic-like tool can be widely used to elucidate dynamic genetic processes in human tissue formation.     

On the Three-Finger Protein Domain Fold and CD59-Like Proteins in Schistosoma mansoni

Background

It is believed that schistosomes evade complement-mediated killing by expressing regulatory proteins on their surface. Recently, six homologues of human CD59, an important inhibitor of the complement system membrane attack complex, were identified in the schistosome genome. Therefore, it is important to investigate whether these molecules could act as CD59-like complement inhibitors in schistosomes as part of an immune evasion strategy.

Methodology/Principal Findings

Herein, we describe the molecular characterization of seven putative SmCD59-like genes and attempt to address the putative biological function of two isoforms. Superimposition analysis of the 3D structure of hCD59 and schistosome sequences revealed that they contain the three-fingered protein domain (TFPD). However, the conserved amino acid residues involved in complement recognition in mammals could not be identified. Real-time RT-PCR and Western blot analysis determined that most of these genes are up-regulated in the transition from free-living cercaria to adult worm stage. Immunolocalization experiments and tegument preparations confirm that at least some of the SmCD59-like proteins are surface-localized; however, significant expression was also detected in internal tissues of adult worms. Finally, the involvement of two SmCD59 proteins in complement inhibition was evaluated by three different approaches: (i) a hemolytic assay using recombinant soluble forms expressed in Pichia pastoris and E. coli; (ii) complement-resistance of CHO cells expressing the respective membrane-anchored proteins; and (iii) the complement killing of schistosomula after gene suppression by RNAi. Our data indicated that these proteins are not involved in the regulation of complement activation.

Conclusions

Our results suggest that this group of proteins belongs to the TFPD superfamily. Their expression is associated to intra-host stages, present in the tegument surface, and also in intra-parasite tissues. Three distinct approaches using SmCD59 proteins to inhibit complement strongly suggested that these proteins are not complement inhibitors and their function in schistosomes remains to be determined.     

Resveratrol Possesses Protective Effects in a Pristane-Induced Lupus Mouse Model

Background

Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease characterized by the production of autoantibodies. To date, no therapy has been found to satisfactorily treat SLE. SIRT1 deficiency results in the development of an autoimmune syndrome in mice, including a high titer of anti-nuclear antibody in serum, immunoglobulin deposition in the kidney, and immune complex glomerulonephritis. Resveratrol is an activator of SIRT1 and possesses anti-inflammation and immune-regulatory properties.

Objective

To evaluate the preventative effects of resveratrol on a pristane-induced lupus animal model and assess its putative immune modulation effects.

Methods

BALB/c mice received a single intraperitoneal injection of 0.5 ml of pristane on day 1 and then various doses of resveratrol were given to the mice daily starting on day 2 and continuing for seven months. The autoantibodies in serum and supernatants were measured. Single cells isolated from spleen, isolated CD4+ T cells, and CD19+ B cells were cultured with or without resveratrol in vitro and assessed by flow cytometry.

Results

Resveratrol attenuated proteinuria, immunoglobuin depositon in kidney, and glomerulonephritis as well as IgG1 and IgG2a in serum in pristane-induced lupus mice. Resveratrol also suppressed CD69 and CD71 expression on CD4+ T cells as well as CD4+ T cell proliferation, induced CD4+ T cell apoptosis, and decreased CD4 IFNγ⁺ Th1 cells and the ratio of Th1/Th2 cells in vitro. In vitro antibody production and proliferation of B cells were also inhibited.

Conclusion

Resveratrol possesses protective effects in pristane-induced lupus mice and may represent a novel approach for the management of SLE.     

Inactivation of MARCH5 Prevents Mitochondrial Fragmentation and Interferes with Cell Death in a Neuronal Cell Model

Purpose

To study the impact of the mitochondrial ubiquitin ligase MARCH5 on mitochondrial morphology and induction of apoptosis using an in vitro model of neuronal precursor cells exposed to glaucoma-relevant stress conditions.

Methods

RGC5 cells transfected with expression constructs for MARCH5, MARCH5^H43W, Dpr1^K38A or vector control were exposed to either elevated pressure of 30 mmHg, oxidative stress caused by mitochondrial electron transport chain (ETC) inhibition, or hypoxia-reoxygenation conditions. Mitochondrial morphology of RGC5 cells was analyzed following staining of the mitochondrial marker cytochrome c and photoactivatable GFP (PAGFP) diffusion assay. Induction of apoptotic cell death in these cells was determined by analyzing the release of cytochrome c from mitochondria into the cytosol and flow cytometry.

Results

Exposure of RGC5 cells to oxidative stress conditions as well as to elevated pressure resulted in the fragmentation of the mitochondrial network in control cells as well as in cells expressing MARCH5. In cells expressing inactive MARCH5^H43W or inactive Drp^K38A, mitochondrial fragmentation was significantly blocked and mitochondrial morphology was comparable to that of control cells under normal conditions. Exposure of RGC5 cells to elevated pressure or oxidative stress conditions induced apoptotic cell death as assessed by cytochrome c release and DNA staining, while expression of dominant-negative MARCH5^H43W or Drp1^K38A did significantly delay cell death.

Conclusion

Preventing mitochondrial fragmentation through interference with the mitochondrial fission machinery protects neuronal cells from programmed cell death following exposure to stressors physiologically relevant to the pathogenesis of glaucoma.