Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum.

Localization of human MHC class I-restricted T cell epitopes in the circumsporozoite (CS) protein of the human parasite Plasmodium falciparum is an important objective in the development of antimalarial vaccines. To this purpose, we synthesized a series of overlapping synthetic 20-mer peptides, spanning the entire sequence of the 7G8 CS molecule except for the central repeat B cell domain. The P.f.CS peptides were first tested for their ability to bind to the human MHC class I HLA-A2.1 molecule on T2, a human cell line. Subsequently, the use of a series of shorter peptide analogues allowed us to determine the optimal A2.1 binding sequence present in several of the 20-mers. Binding P.f.CS peptides were further tested for their capacity to activate PBL from HLA-A2.1+ immune donors living in a malaria-endemic area. Specific IFN-gamma production was detected in the supernatant of cultures of PBL from exposed individuals. Cytotoxic T cell lines and clones were derived from the PBL of one responder, and their activity was shown to be HLA-A2.1-restricted and specific for the peptide 334-342 of the CS protein. In addition, double transgenic HLA-A2.1 x human beta 2-microglobulin mice were immunized with peptide 1-10 of the CS protein. T cells derived from immune lymph nodes displayed a peptide-specific HLA-A2.1-restricted cytolytic activity after one in vitro stimulation.

Proteomic analysis of Intercept-treated platelets.

In the past decades, transfusion medicine has been driven by the quest for increased safety against transfusion-transmitted infections, mainly by better donor selection and by the development of improved serological and nucleic-acid-based screening assays. Recently, pathogen reduction technologies became available and started to be implemented in several countries, with the primary goal to fight against bacterial contamination of blood products, a rare but dramatic event against which there was no definitive measure. Though pathogen reduction technologies represent a quantum leap in transfusion safety, the biomedical efficacy of platelet concentrates (PCs) treated with various pathogen reduction techniques has been recently questioned by clinical studies. Here, a gel-based proteomic analysis of PCs (n=5), Intercept-treated or untreated, from pooled buffy-coat (10 donors per PC) at Days 1, 2 and 8, shows that the Intercept process that is the most widespread pathogen reduction technique to date, has relatively low impact on the proteome of treated platelets: the process induces modifications of DJ-1 protein, glutaredoxin 5, and G(i)alpha 2 protein. As for the impact of storage, chloride intracellular channel protein 4 (CLIC4) and actin increased independently of Intercept treatment during storage. Whereas alteration of the DJ-1 protein and glutaredoxin 5 points out an oxidative stress-associated lesion, modification of G(i)alpha2 directly connects a possible Intercept-associated lesion to haemostatic properties of Intercept-treated platelets. This article is part of a Special Issue entitled: Integrated omics.

Association between mannose-binding lectin (MBL) deficiency and cytomegalovirus (CMV) infection after kidney transplantation

MBLdeficiency is thought to be a risk factor for the development of viral infection, such as genital herpes and HSV-2 meningitis. However, there is limited data on the possible interaction between MBL and CMV, especially after organ transplantation. Between 2003 and 2005, we measured MBL levels in 16 kidney transplant recipients with high-risk CMV serostatus (donor positive/recipient negative, D+/R−). All patients receivedCMV prophylaxis of valganciclovir 450 mg/day for 3 months after transplantation. After stopping valganciclovir, CMV-DNA was measured in whole blood by real time PCR every 2 weeks for 3 months. CMV infections were diagnosed according to the recommendations of the AST. MBL levels were measured in stored pre-transplantation sera by an investigator blinded to the CMV complications. MBL levels below 500 ng/ml were considered as being functionally deficient. After a follow-up of at least 10 months, seven patients out of 16 developed CMV disease (three CMV syndrome, and four probable invasive disease, i.e. two colitis and two hepatitis), four patients developed asymptomatic CMV infection, and five patients never developed any sign of CMV replication. Peak CMV-DNA was higher in patients with CMV disease than in those with asymptomatic infection (4.64 versus 2.72 mean log copy CMV-DNA/106 leukocytes, p < 0.05). Overall, 9/16 patients (56%) had MBL deficiency: 5/7 (71%) of patients with CMV disease, 4/4 (100%) of patients with asymptomatic CMVinfection, and 0/5 (0%) of patients withoutCMVinfection (p < 0.005, between CMV infection/disease versus no infection or control blood donors). There were no significant differences in age, gender or immunosuppressive regimens between the groups. MBL deficiency may be a significant risk factor for the development of post-prophylaxisCMVinfection in D+/R−kidney recipients, suggesting a new role of innate immunity in the control of CMV infection after organ transplantation.

Role of intra-operative duplex ultrasound in living renal donor transplantation

Objective: The treatment of choice in end-stage renal disease is¦transplantation.¦Hemodynamic disturbances can evoke graft loss, while early ultrasound¦identification of vascular problems improves outcome. The primary endpoint¦of this study was to identify differences in post-op complications with and¦without systematic, intraoperative Doppler ultrasound use. A secondary aim¦was to find a predictive resistance index limit which would show where surgical¦reintervention was necessary.¦Methods: Between Jan 2000 and Dec 2010, 108 renal transplants were¦performed from living donors at our institution. In group 1 (n = 67),¦intra-operative duplex ultrasound and intra-parenchymatous resistance index¦measurements assessed patients, while in group 2 (n = 41), no ultrasound was¦performed.¦Results. There were no inter-group differences in the overall post-op¦complication rate or in benefit to graft or patient survival with Doppler use,¦however, significantly more vascular complications (10% vs. 0%, p = 0·02) and¦more acute rejections (37% vs. 10%) occurred in group 2 than in group 1,¦respectively. When the resistance index was <0·5 intra-operatively, immediate¦surgical revision was undertaken to raise the index >0·6.¦Results: There were no inter-group differences in the overall post-op¦complication rate or in benefit to graft or patient survival with Doppler use,¦however, significantly more vascular complications (10% vs. 0%, p = 0·02) and¦more acute rejections (37% vs. 10%) occurred in group 2 than in group 1,¦respectively. When the resistance index was <0·5 intra-operatively, immediate¦surgical revision was undertaken to raise the index >0·6.¦Conclusion: This is the first report demonstrating benefits of systematic¦intraoperative Doppler ultrasound on post-operative complications in renal¦transplantation from living donors. Our results support surgical revision with a¦resistance index <0·5.

Impact of HLA A2 and cytomegalovirus serostatus on outcomes in patients with leukemia following matched-sibling myeloablative allogeneic hematopoietic cell transplantation.

BACKGROUND AND OBJECTIVES: Donor cytomegalovirus seropositivity was reported to improve leukemia outcomes in HLA-A2 identical hematopoietic cell transplant (HCT) recipients, due to a possible cross-reactivity of donor HLA-A2-restricted CMV-specific T cells with minor histocompatibility (H) antigen of recipient cells. This study analyzed the role of donor CMV serostatus and HLA-A2 status on leukemia outcomes in a large population of HLA-identical HCT recipients. DESIGN AND METHODS: Leukemia patients transplanted between 1992 and 2003 at the Fred Hutchinson Cancer Research Center were categorized as standard risk [leukemia first remission, chronic myeloid leukemia in chronic phase (CML-CP)] and high risk (advanced disease) patients. Time-to-event analysis was used to evaluate the risk of relapse and death associated with HLA-A2 status and donor CMV serostatus. RESULTS: In standard risk patients, acute leukemia (p<0.001) and sex mismatch (female to male, p=0.004)) independently increased the risk of death, while acute leukemia increased the risk of relapse (p<0.001). In high risk patients acute leukemia (p=0.01), recipient age > or = 40 (p=0.005) and herpes simplex virus (HSV) seropositivity (p<0.001) significantly increased the risk death; HSV seropositivity (p=0.006) increased the risk of relapse. Donor CMV serostatus had no significant effect on mortality or relapse in any HLA group. INTERPRETATION AND CONCLUSION: This epidemiological study did not confirm the previously reported effect of donor CMV serostatus on the outcomes of leukemia in HLA-A2-identical HCT recipients. Addressing the question of cross-reactivity of HLA-A2-restricted CMV-specific T cells with minor H antigens in a clinical study would require knowledge of the patient's minor H antigen genotype. However, because of the unbalanced distribution of HLA-A2-restricted minor H antigens in the population and their incomplete identification, this question might be more appropriately evaluated in in vitro experiments than in a clinical study.

Hepatitis G virus infection in primary Sjögren's syndrome: analysis in a series of 100 patients.

OBJECTIVE To determine the prevalence and clinical significance of hepatitis G virus (HGV) infection in a large cohort of patients with primary Sjögren¿s syndrome (SS). PATIENTS AND METHODS The study included 100 consecutive patients (92 female and eight male), with a mean age of 62 years (range 31¿80) that were prospectively visited in our unit. All patients fulfilled the European Community criteria for SS and underwent a complete history, physical examination, as well as biochemical and immunological evaluation for liver disease. Two hundred volunteer blood donors were also studied. The presence of HGV-RNA was investigated in the serum of all patients and donors. Aditionally, HBsAg and antibodies to hepatitis C virus were determined. RESULTS Four patients (4%) and six volunteer blood donors (3%) presented HGV-RNA sequences in serum. HGV infection was associated with biochemical signs of liver involvement in two (50%) patients. When compared with primary SS patients without HGV infection, no significant differences were found in terms of clinical or immunological features. HCV coinfection occurs in one (25%) of the four patients with HGV infection. CONCLUSION The prevalence of HGV infection in patients with primary SS is low in the geographical area of the study and HCV coinfection is very uncommon. HGV infection alone does not seen to be an important cause of chronic liver injury in the patients with primary SS in this area.

A recombinant rubella virus E1 glycoprotein as a rubella vaccine candidate.

A recombinant rubella virus E1 (rE1) glycoprotein was produced and some of its chemical and immunological features were characterized. Two animal models were then used to establish that the rE1 glycoprotein and rubella virus particles shared antigenic and immunogenic properties. In the first one, sera from rE1 glycoprotein-immunized BALB/c mice neutralized in vitro rubella virus infection. In the second model, severe combined immune deficient (SCID) mice implanted with tonsil fragments from rubella immune donors and immunized with rE1 glycoprotein produced human anti-rubella virus antibodies. Altogether, these results showed that immunization with rE1 glycoprotein elicited neutralizing anti-rubella virus antibodies. This study thus indicated that the rE1 glycoprotein could constitute a non-replicating rubella vaccine.

Low molecular weight carboxylic acids in oxidizing porphyry copper tailings

The distribution of low molecular weight carboxylic acids (LMWCA) was investigated in pore water profiles from two porphyry copper tailings impoundments in Chile (Piuquenes at La Andina and Cauquenes at El Teniente mine). The objectives of this study were (1) to determine the distribution of LMWCA, which are interpreted to be the metabolic byproducts of the autotroph microbial community in this low organic carbon system, and (2) to infer the potential role of these acids in cycling of Fe and other elements in the tailings impoundments. The speciation and mobility of iron, and potential for the release of H+ via hydrolysis of the ferric iron, are key factors in the formation of acid mine drainage in sulfidic mine wastes. In the low-pH oxidation zone of the Piuquenes tailings, Fe(III) is the dominant iron species and shows high mobility. LMWCA, which occur mainly between the oxidation front down to 300 cm below the tailings surface at both locations (e.g., max concentrations of 0.12 mmol/L formate, 0.17 mmol/L acetate, and 0.01 mmol/L pyruvate at Piuquenes and 0.14 mmol/L formate, 0.14 mmol/L acetate, and 0.006 mmol/L pyruvate at Cauquenes), are observed at the same location as high Fe concentrations (up to 71.2 mmol/L Fe(II) and 16.1 mmol/L Fe(III), respectively). In this zone, secondary Fe(111) hydroxides are depleted. Our data suggest that LMWCA may influence the mobility of iron in two ways. First, complexation of Fe(III), through formation of bidentate Fe(III)-LMWCA complexes (e.g., pyruvate, oxalate), may enhance the dissolution of Fe(III) (oxy)hydroxides or may prevent precipitation of Fe(III) (oxy)hydroxides. Soluble Fe(III) chelate complexes which may be mobilized downward and convert to Fe(II) by Fe(III) reducing bacteria. Second, monodentate LMWCA (e.g., acetate and formate) can be used by iron-reducing bacteria as electron donors (e.g., Acidophilum spp.), with ferric iron as the electron acceptor. These processes may, in part, explain the low abundances of secondary Fe(III) hydroxide precipitates below the oxidation front and the high concentrations of Fe(II) observed in the pore waters of some low-sulfide systems. The reduction of Fe(III) and the subsequent increase of iron mobility and potential acidity transfer (Fe(II) oxidation can result in the release of H+ in an oxic environment) should be taken in account in mine waste management strategies.

Analysis of the alternative pathways for the beta-oxidation of unsaturated fatty acids using transgenic plants synthesizing polyhydroxyalkanoates in peroxisomes.

Degradation of fatty acids having cis-double bonds on even-numbered carbons requires the presence of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. Two alternative pathways have been described to degrade these fatty acids. One pathway involves the participation of the enzymes 2, 4-dienoyl-coenzyme A (CoA) reductase and Delta(3)-Delta(2)-enoyl-CoA isomerase, whereas the second involves the epimerization of R-3-hydroxyacyl-CoA via a 3-hydroxyacyl-CoA epimerase or the action of two stereo-specific enoyl-CoA hydratases. Although degradation of these fatty acids in bacteria and mammalian peroxisomes was shown to involve mainly the reductase-isomerase pathway, previous analysis of the relative activity of the enoyl-CoA hydratase II (also called R-3-hydroxyacyl-CoA hydro-lyase) and 2,4-dienoyl-CoA reductase in plants indicated that degradation occurred mainly through the epimerase pathway. We have examined the implication of both pathways in transgenic Arabidopsis expressing the polyhydroxyalkanoate synthase from Pseudomonas aeruginosa in peroxisomes and producing polyhydroxyalkanoate from the 3-hydroxyacyl-CoA intermediates of the beta-oxidation cycle. Analysis of the polyhydroxyalkanoate synthesized in plants grown in media containing cis-10-heptadecenoic or cis-10-pentadecenoic acids revealed a significant contribution of both the reductase-isomerase and epimerase pathways to the degradation of these fatty acids.

Sandwich enzyme immunoassay using three monoclonal antibodies against different epitopes of carcinoembryonic antigen (CEA).

Purified monoclonal antibodies (Mab) produced by 3 hybridomas and reacting with 3 different epitopes of carcinoembryonic antigen (CEA) were used in a solid phase enzyme immunoassay. Two Mabs were physically adsorbed to polystyrene balls and the third Mab was coupled to alkaline phosphatase using the bifunctional reagent N-succinimidyl-3-(2-pyridyldithio)-propionate. During a first incubation, CEA from heat-extracted serum samples was immunoadsorbed to the antibody coated balls. After washing of the balls, bound CEA was detected by a second incubation with the enzyme coupled Mab. The sensitivity of the assay was 0.6 ng per ml of serum. A total of 196 serum samples from patients with various types of carcinoma, with liver cirrhosis, or from healthy blood donors with or without smoking habits, were tested. The results obtained with the monoclonal enzyme immunoassay (M-EIA) were compared with those obtained with perchloric acid extracts of the same serum samples tested by an inhibition radioimmunoassay using conventional goat anti-CEA antiserum. There was an excellent correlation between the two assays. In particular, the new M-EIA gave good results for the detection of tumor recurrences in the follow-up of colon carcinoma patients. However, despite the use of exclusively monoclonal antibodies the new assay detected a similar percentage of slightly elevated CEA values as the conventional assay in patients with non-malignant disease, suggesting that the CEA associated with non-malignant diseases is immunologically identical to the CEA released by colon carcinoma.

Urological complications in renal transplantation from cadaveric donor grafts: a retrospective analysis of 20 years.

INTRODUCTION: This study is a retrospective analysis of ureteral complications and their management from a monocenter series of 277 consecutive renal transplantations. MATERIALS AND METHODS: From September 1979 to June 1999, 277 renal transplantations (cadaveric origin) were performed in 241 patients. The ureter from the kidney graft was inserted into the bladder according to the technique of extravesical implantation described by Lich-Gregoir and Campos-Freire. The study analyzed the time of occurrence and the type of complications observed. The different procedures to restore the transplanted urinary tract are presented. RESULTS: Complications occurred in 43/277 renal transplantations (15.5%). Anastomotic urine leakage or ureteral stricture were the most frequent. The time to appearance of these complications was either short (<1 month) or late (>1 month) in a similar number of cases. Most cases were managed surgically: 33/43 cases (76.7%). The most frequent surgical repair was ureterovesical reimplantation (n=13), followed by: ureteroureteral end-to-end anastomosis (native ureter-ureter transplant, n=5); pyeloureteral anastomosis (native ureter-renal pelvis transplant, n=5); simple revision of ureterovesical implantation (n=4); resection and end-to-end anastomosis of the transplant ureter (n=2); calico-vesicostomy (graft-bladder, n=1); implantation according to Boari (n=1); pyelovesicostomy with bipartition of bladder (n=1), and pyeloileocystoplasty with detubularized ileal graft (n=1). No deaths related to any of the urological complications were reported. However, 2 consecutive vesico-renal refluxes led to the loss of the kidney graft in the long-term. CONCLUSION: The rate of complications observed in this retrospective analysis is similar to the experience of other studies, ranging from 2 to 20%. If the classical extravesical ureteral bladder implantation is to remain an attractive technique due to its simplicity, the surgical team at the training center should be aware of all the means to prevent any ureteral complications, such as the choice of another implantation technique and/or insertion of a transient ureteral stent.

Nitric Oxide Induces the Expression of the Monocarboxylate Transporter MCT4 in Cultured Astrocytes by a cGMP-Independent Transcriptional Activation

The monocarboxylate transporter MCT4 is a proton-linked carrier particularly important for lactate release from highly glycolytic cells. In the central nervous system, MCT4 is exclusively expressed by astrocytes. Surprisingly, MCT4 expression in primary cultures of mouse cortical astrocytes is conspicuously low, suggesting that an external, nonastrocytic signal is necessary to obtain the observed pattern of expression in vivo. Here, we demonstrate that nitric oxide (NO), delivered by various NO donors, time- and dose-dependently induces MCT4 expression in cultured cortical astrocytes both at the mRNA and protein levels. In contrast, NO does not enhance the expression of MCT1, the other astrocytic monocarboxylate transporter. The transcriptional effect of NO is not mediated by a cGMP-dependent mechanism as shown by the absence of effect of a cGMP analog or of a selective guanylate cyclase inhibitor. NO causes an increase in astrocytic lactate transport capacity which requires the enhancement of MCT4 expression as both are prevented by the use of a specific siRNA against MCT4. In addition, cumulated lactate release by astrocytes over a period of 24 h was also enhanced by NO treatment. Our data suggest that NO represents a putative intercellular signal to control MCT4 expression in astrocytes and in doing so, to facilitate lactate transfer to other surrounding cell types in the central nervous system. (C) 2011 Wiley-Liss, Inc.

Macular and retinal dysfunction of unknown origin in adults with normal fundi: evidence for an autoimmune pathophysiology.

Here we report the discovery of and phenotypic characterization of a retinal disorder of unknown origin in adults using clinical, electrophysiological and psychophysical techniques, and to seek the presence of circulating retinal autoantibodies in the sera of these patients. Sixteen patients were identified with progressive bilateral visual loss over a period of months. Ten of the patients were male, and the average age was 55.3 years (range from 43 to 76 years). Known causes such as carcinoma-associated retinopathy, acute zonal occult outer retinopathy and hereditary cone dystrophy appeared unlikely. Investigations included electrophysiology, fundus autofluorescence imaging and psychophysical tests. The sera of these patients were analyzed with indirect immunocytochemistry and Western immunoblot analysis on murine (BALB/c) retinal tissue for the presence of retinal autoantibodies. Bilateral visual loss and photophobia progressed over a period of months to years (average 28.7 months, range 3-67) and subsequently stabilized. No abnormality was observed by biomicroscopy, angiography or autofluorescence imaging. Electrophysiology indicated predominant cone-system dysfunction, either macular or generalized, and post-phototransduction involvement in 9 patients (56%). Photopic and scotopic visual fields and dark adaptation kinetics showed both cone and rod system involvement in all cases. Heterogeneous immunohistochemical staining patterns were seen with the sera of these patients as compared with controls. A majority of the affected patients (9/15) stained with an antinuclear pattern. The retinal autoantibodies from the sera of most patients reacted with the retinal proteins of molecular weight between 34 and 40 kDa. The aetiology of this distinctive retinal disorder therefore appears to be mediated through an autoimmune mechanism.