Time-Course In Vivo Trafficking Pattern and Effector Function of Donor-Specific Regulatory T Cells in Response to an Allograft.

Background: Experimental data have suggested that adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs), capable of controlling immune responses to specifi c auto- or alloantigens, could be used as a therapeutic strategy to promote specifi c tolerance in T-cell mediated diseases and in organ transplantation (Tx). However, before advocating the application of immunotherapy with Tregs in Tx, we need to improve our understanding of their in vivo homeostasis, traffi cking pattern and effector function in response to alloantigens. Methods : Donor-antigen specifi c murine Tregs were generated and characterized in vitro following our described protocols. Using an adoptive transfer and skin allotransplantation model, we have analyzed the in vivo expansion and homing of fl uorescent-labeled effector T cells (Teff) and Tregs, at different time-points after Tx, using fl ow-cytometry as well as fl uorescence microscopy techniques. Results: Tregs expressed CD62L, CCR7 and CD103 allowing their homing into lymphoid and non-lymphoid tissues (gut, skin) after intravenous injection. While hyporesponsive to TCR stimulation in vitro, transferred Tregs survived, migrated to secondary lymphoid organs and preferentially expanded within the allograft draining lymph nodes. Furthermore, Foxp3+ cells could be detected inside the allograft as early as day 3-5 after Tx. At a much later time-point (day 60 after Tx), graft-infi ltrating Foxp3+ cells were also detectable in tolerant recipients. When transferred alone, CD4+CD25- Teff cells expanded within secondary lymphoid organs and infi ltrated the allograft by day 3-5 after Tx. The co-transfer of Tregs limited the expansion of alloreactive Teff cells as well as their recruitment into the allograft. The promotion of graft survival observed in the presence of Tregs was in part mediated by the inhibition of the production of effector cytokines by CD4+CD25- T cells. Conclusion: Taken together, our results suggest that the suppression of allograft rejection and the induction of Tx tolerance are in part dependant on the alloantigendriven homing and expansion of Tregs. Thus, the appropriate localization of Tregs may be critical for their suppressive function in vivo.

Mineralocorticoid receptor is involved in rat and human ocular chorioretinopathy.

Central serous chorioretinopathy (CSCR) is a vision-threatening eye disease with no validated treatment and unknown pathogeny. In CSCR, dilation and leakage of choroid vessels underneath the retina cause subretinal fluid accumulation and retinal detachment. Because glucocorticoids induce and aggravate CSCR and are known to bind to the mineralocorticoid receptor (MR), CSCR may be related to inappropriate MR activation. Our aim was to assess the effect of MR activation on rat choroidal vasculature and translate the results to CSCR patients. Intravitreous injection of the glucocorticoid corticosterone in rat eyes induced choroidal enlargement. Aldosterone, a specific MR activator, elicited the same effect, producing choroid vessel dilation -and leakage. We identified an underlying mechanism of this effect: aldosterone upregulated the endothelial vasodilatory K channel KCa2.3. Its blockade prevented aldosterone-induced thickening. To translate these findings, we treated 2 patients with chronic nonresolved CSCR with oral eplerenone, a specific MR antagonist, for 5 weeks, and observed impressive and rapid resolution of retinal detachment and choroidal vasodilation as well as improved visual acuity. The benefit was maintained 5 months after eplerenone withdrawal. Our results identify MR signaling as a pathway controlling choroidal vascular bed relaxation and provide a pathogenic link with human CSCR, which suggests that blockade of MR could be used therapeutically to reverse choroid vasculopathy.

DETERMINATION OF CELL-SPECIFIC NEUROTOXICITY OF MALONATE, METHYLMALONATE AND PROPIONATE IN A 3D RAT BRAIN CELL AGGREGATE SYSTEM

Malonate, methylmalonate and propionate are potentially neurotoxic metabolites in branched-chain organic acidurias. Their effects were tested on cultured 3D rat brain cell aggregates, using dosages of 0.1, 1.0 and 10.0 mM with a short but intense (twice a day over 3 days) and a longer but less intense treatment (every 3 rdday over 9 days). CNS cell-specific immunohistochemical stainings allowed the follow-up of neurons (axons, phosphorylated medium-weight neurofilament), astrocytes (glial fibrillary acidic protein) and oligodendrocytes (myelin basic protein). Methylmalonate and malonate were quantified by tandem mass spectrometry. Tandem mass spectrometry analysis of harvested brain cell aggregates revealed clear intracellular accumulation of methylmalonate and malonate. In immunohistochemical stainings oligodendrocytes appeared the most affected brain cells. The MBP signal disappeared already at 0.1 mM treatment with each metabolite. Mature astrocytes were not affected by propionate, while immature astrocytes on intense treatment with propionate developed cell swelling. 1 mM methylmalonate induced cell swelling of both immature and mature astrocytes , while 1 mM malonate only affected mature astrocytes. Neurons were not affected by methylmalonate, but 10.0 mM malonate on less intense treatment and 0.1, 1.0 and 10.0 mM propionate on intense treatment affected axonal growth. Our study shows significant uptake and deleterious effects of these metabolites on brain cells, principally on astrocytes and oligodendrocytes. This may be explained by the absence of the pathway in glial cells, which thus are not able to degrade these metabolites. Further studies are ongoing to elucidate the underlying mechanisms of the observed neurotoxic effects.

An oligonucleotide probe derived from kDNA minirepeats is specific for Leishmania (Viannia)

Sequence analysis of Leishmania (Viannia) kDNA minicircles and analysis of multiple sequence alignments of the conserved region (minirepeats) of five distinct minicircles from L. (V.) braziliensis species with corresponding sequences derived from other dermotropic leishmanias indicated the presence of a sub-genus specific sequence. An oligonucleotide bearing this sequence was designed and used as a molecular probe, being able to recognize solely the sub-genus Viannia species in hybridization experiments. A dendrogram reflecting the homologies among the minirepeat sequences was constructed. Sequence clustering was obtained corresponding to the traditional classification based on similarity of biochemical, biological and parasitological characteristics of these Leishmania species, distinguishing the Old World dermotropic leishmanias, the New World dermotropic leishmanias of the sub-genus Leishmania and of the sub-genus Viannia.

Physicians' attitudes towards prevention: importance of intervention-specific barriers and physicians' health habits.

BACKGROUND: Several studies have explored physicians' attitudes towards prevention and barriers to the delivery of preventive health interventions. However, the relative importance of these previously identified barriers, both in general terms and in the context of a number of specific preventive interventions, has not been identified. Certain barriers may only pertain to a subset of preventive interventions. OBJECTIVES: We aimed to determine the relative importance of identified barriers to preventive interventions and to explore the association between physicians' characteristics and their attitudes towards prevention. METHODS: We conducted a cross-sectional survey of 496 of the 686 (72.3% response rate) generalist physicians from three Swiss cantons through a questionnaire asking physicians to rate the general importance of eight preventive health strategies and the relative importance of seven commonly cited barriers in relation to each specific preventive health strategy. RESULTS: The proportion of physicians rating each preventive intervention as being important varied from 76% for colorectal cancer screening to 100% for blood pressure control. Lack of time and lack of patient interest were generally considered to be important barriers by 41% and 44% of physicians, respectively, but the importance of these two barriers tended to be specifically higher for counselling-based interventions. Lack of training was most notably a barrier to counselling about alcohol and nutrition. Four characteristics of physicians predicted negative attitudes toward alcohol and smoking counselling: consumption of more than three alcoholic drinks per day [odds ratio (OR) = 8.4], sedentary lifestyle (OR = 3.4), lack of national certification (OR = 2.2) and lack of awareness of their own blood pressure (OR = 2.0). CONCLUSIONS: The relative importance of specific barriers varies across preventive interventions. This points to a need for tailored practice interventions targeting the specific barriers that impede a given preventive service. The negative influence of physicians' own health behaviours indicates a need for associated population-based interventions that reduce the prevalence of high-risk behaviours in the population as a whole.

Naturally acquired MAGE-A10- and SSX-2-specific CD8+ T cell responses in patients with hepatocellular carcinoma.

Immunotherapy is being proposed to treat patients with hepatocellular carcinoma (HCC). However, more detailed knowledge on tumor Ag expression and specific immune cells is required for the preparation of highly targeted vaccines. HCC express a variety of tumor-specific Ags, raising the question whether CTL specific for such Ags exist in HCC patients. Indeed, a recent study revealed CTLs specific for two cancer-testis (CT) Ags (MAGE-A1 and MAGE-A3) in tumor infiltrating lymphocytes of HCC patients. Here we assessed the presence of T cells specific for additional CT Ags: MAGE-A10, SSX-2, NY-ESO-1, and LAGE-1, which are naturally immunogenic as demonstrated in HLA-A2(+) melanoma patients. In two of six HLA-A2(+) HCC patients, we found that MAGE-A10- and/or SSX-2-specific CD8(+) T cells naturally responded to the disease, because they were enriched in tumor lesions but not in nontumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, providing evidence that these CTL were selected in vivo for high avidity Ag recognition. Therefore, besides melanoma, HCC is the second solid human tumor with clear evidence for in vivo tumor recognition by T cells, providing the rational for specific immunotherapy, based on immunization with CT Ags such as MAGE-A10 and SSX-2.

Site-specific conjugation of a radioiodinated phenethylamine derivative to a monoclonal antibody results in increased radioactivity localization in tumor.

The preparation of a novel radioiodination reagent, the (aminooxy)acetyl derivative of (p-[125]-iodophenyl)ethylamine, is described. Conventional radioiodination of proteins involves the formation of iodotyrosine residues, but for in vivo applications such as thyroid or stomach immunoscintigraphy, the susceptibility of these residues to tissue dehalogenases constitutes a serious disadvantage. Using our new compound, which has a particularly nonreactive aromatic ring, we confirm and extend studies published by other workers indicating the much greater in vivo stability of iodophenyl compounds compared to the more conventional iodophenolic ones. In addition, the aminooxy group of our reagent gives a stable and specific linkage to aldehyde groups formed by periodate oxidation on the sugar moiety of antibody molecules. In vitro, favorable binding activity and high stability was obtained with a (([125I]iodoaryl)amino)oxy labeled monoclonal antibody directed against carcinoembryonic antigen. In vivo, using paired labeling experiments in nude mice bearing colon carcinoma xenografts, the (([125I]iodoaryl)amino)oxy-MAb (MAb = monoclonal antibody) was compared with the same MAb 131I-labeled by conventional chloramine-T method. Tumor 125I concentration of (arylamino)oxy MAb (measured as percent injected dose per gram) was significantly higher as compared to values obtained with a conventionally labeled 131I antibody. Additionally, thyroid uptake, an indicator of iodine release from the antibody, was up to 25 times lower after injection of 125I-MAb obtained by the new method as compared to the conventionally iodinated 131I-MAb.

Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells.

New anti-cancer agents are being developed that specifically recognise tumour cells. Recognition is dependent upon the enhanced expression of antigenic determinants on the surface of tumour cells. The tumour exposure and the extracellular accessibility of the mucin MUC-1 make this marker a suitable target for tumour diagnosis and therapy. We isolated and characterised six human scFv antibody fragments that bound to the MUC-1 core protein, by selecting a large naive human phage display library directly on a MUC-1-expressing breast carcinoma cell line. Their binding characteristics have been studied by ELISA, FACS and indirect immunofluorescence. The human scFv antibody fragments were specific for the tandem repeat region of MUC-1 and their binding is inhibited by soluble antigen. Four human scFv antibody fragments (M2, M3, M8, M12) recognised the hydrophilic PDTRP region of the MUC-1 core protein, which is thought to be an immunodominant region. The human scFv antibody fragments were stable in human serum at 37 degrees C and retained their binding specificity. For imaging or targeting to tumours over-expressing MUC-1, it might be feasible to use these human scFv, or multivalent derivatives, as vehicles to deliver anti-cancer agents.

Characterization of Endotrypanum Parasites Using Specific Monoclonal Antibodies

A large number of Endotrypanum stocks (representing an heterogeneous population of strains) have been screened against a panel of monoclonal antibodies (MAbs) derived for selected species of Endotrypanum or Leishmania, to see whether this approach could be used to group/differentiate further among these parasites. Using different immunological assay systems, MAbs considered specific for the genus Endotrypanum (E-24, CXXX-3G5-F12) or strain M6159 of E. schaudinni (E-2, CXIV-3C7-F5) reacted variably according to the test used but in the ELISA or immunofluorescence assay both reacted with all the strains tested. Analyses using these MAbs showed antigenic diversity occurring among the Endotrypanum strains, but no qualitative or quantitative reactivity pattern could be consistently related to parasite origin (i.e., host species involved) or geographic area of isolation. Western blot analyses of the parasites showed that these MAbs recognized multiple components. Differences existed either in the epitope density or molecular forms associated with the antigenic determinants and therefore allowed the assignment of the strains to specific antigenic groups. Using immunofluorescence or ELISA assay, clone E-24 produced reaction with L. equatorensis (which is a parasite of sloth and rodent), but not with other trypanosomatids examined. Interestingly, the latter parasite and the Endotrypanum strains cross-reacted with a number of MAbs that were produced against members of the L. major-L. tropica complex

Specific Identification of Biomphalaria tenagophila and Biomphalaria occidentalis Populations by the Low Stringency Polymerase Chain Reaction

Although Biomphalaria occidentalis and B. tenagophila are indistinguishable on the basis of shell morphology and the majority of their genital organs, only the latter is susceptible to infection with Schistosoma mansoni. Thus, the identification of these species is fundamental to epidemiological studies of schistosomiasis. Here we describe a simple and rapid method for differentiating B. tenagophila from B. occidentalis based on low stringency polymerase chain reaction and using a pair of primers specific for the amplification of the 18S rRNA gene. Analysis of the low stringency product profiles of populations of these snails from different geographical regions confirmed this approach as being applicable to the identification of B. tenagophila and B. occidentalis in cases where classical morphology is inconclusive

Use of a renal-specific oral supplement by haemodialysis patients with low protein intake does not increase the need for phosphate binders and may prevent a decline in nutritional status and quality of life.

BACKGROUND: Protein-energy wasting is a frequent and debilitating condition in maintenance dialysis. We randomly tested if an energy-dense, phosphate-restricted, renal-specific oral supplement could maintain adequate nutritional intake and prevent malnutrition in maintenance haemodialysis patients with insufficient intake. METHODS: Eighty-six patients were assigned to a standard care (CTRL) group or were prescribed two 125-ml packs of Renilon 7.5(R) daily for 3 months (SUPP). Dietary intake, serum (S) albumin, prealbumin, protein nitrogen appearance (nPNA), C-reactive protein, subjective global assessment (SGA) and quality of life (QOL) were recorded at baseline and after 3 months. RESULTS: While intention to treat analysis (ITT) did not reveal strong statistically significant changes in dietary intake between groups, per protocol (PP) analysis showed that the SUPP group increased protein (P < 0.01) and energy (P < 0.01) intakes. In contrast, protein and energy intakes further deteriorated in the CTRL group (PP). Although there was no difference in serum albumin and prealbumin changes between groups, in the total population serum albumin and prealbumin changes were positively associated with the increment in protein intake (r = 0.29, P = 0.01 and r = 0.27, P = 0.02, respectively). The SUPP group did not increase phosphate intake, phosphataemia remained unaffected, and the use of phosphate binders remained stable or decreased. The SUPP group exhibited improved SGA and QOL (P < 0.05). CONCLUSION: This study shows that providing maintenance haemodialysis patients with insufficient intake with a renal-specific oral supplement may prevent deterioration in nutritional indices and QOL without increasing the need for phosphate binders.

Cloning and Characterization of a Schistosoma mansoni cDNA Clone with a Specific Antigenic Expression during Development in a Vertebrate Host

Approximately 2.0 x 10 cDNA clones of an Schistosoma mansoni lgt11 cDNA library were screened in duplicate with serum from infected mice corresponding to distinct phases of infection. A cDNA clone (7/1) was isolated and recognized only by seven week serum. The clone was subcloned in pGEX-2T and Western-blot studies showed a specific antigenic expression confirming that only serum from the chronic phase is capable of recognizing this antigen. Dot-hybridization with RNA from different developmental phases of the parasite showed that the corresponding 7/1 RNA is expressed in all phases of parasite development in vertebrate hosts

Optimum in vitro expansion of human antigen-specific CD8 T cells for adoptive transfer therapy.

Increasing evidence suggests that adoptive transfer of antigen-specific CD8(+) T cells could represent an effective strategy in the fight against chronic viral infections and malignancies such as melanoma. None the less, a major limitation in the implementation of such therapy resides in the difficulties associated with achieving rapid and efficient expansion of functional T cells in culture necessary to obtain the large numbers required for intravenous infusion. Recently, the critical role of the cytokines interleukin (IL)-2, IL-7 and IL-15 in driving T cell proliferation has been emphasized, thus suggesting their use in the optimization of expansion protocols. We have used major histocompatibility complex (MHC) class I/peptide multimers to monitor the expansion of antigen-specific CD8 T lymphocytes from whole blood, exploring the effect of antigenic peptide dose, IL-2, IL-7 and IL-15 concentrations on the magnitude and functional characteristics of the antigen-specific CD8(+) T cells generated. We show here that significant expansions of antigen-specific T cells, up to 50% of the CD8(+) T cell population, can be obtained after a single round of antigen/cytokine (IL-2 or IL-15) stimulation, and that these cells display good cytolytic and interferon (IFN)-gamma secretion capabilities. Our results provide an important basis for the rapid in vitro expansion of autologous T cells from the circulating lymphocyte pool using a simple procedure, which is necessary for the development of adoptive transfer therapies.

An exploratory study on facial emotion recognition capacity in beginning Alzheimer's disease.

Objective: It was the aim of this study to investigate facial emotion recognition (FER) in the elderly with cognitive impairment. Method: Twelve patients with Alzheimer's disease (AD) and 12 healthy control subjects were asked to name dynamic or static pictures of basic facial emotions using the Multimodal Emotion Recognition Test and to assess the degree of their difficulty in the recognition task, while their electrodermal conductance was registered as an unconscious processing measure. Results: AD patients had lower objective recognition performances for disgust and fear, but only disgust was accompanied by decreased subjective FER in AD patients. The electrodermal response was similar in all groups. No significant effect of dynamic versus static emotion presentation on FER was found. Conclusion: Selective impairment in recognizing facial expressions of disgust and fear may indicate a nonlinear decline in FER capacity with increasing cognitive impairment and result from progressive though specific damage to neural structures engaged in emotional processing and facial emotion identification. Although our results suggest unchanged unconscious FER processing with increasing cognitive impairment, further investigations on unconscious FER and self-awareness of FER capacity in neurodegenerative disorders are required.