Comparison of C4d detection on erythrocytes and PTC-C4d to histological signs of antibody-mediated rejection in kidney transplantation.

C4d on erythrocytes (EC4d), C4d peritubular capillary deposition (PTC-C4d) staining and histology were compared in a cross-sectional cohort of 146 renal allograft biopsies (132 patients). EC4d levels paralleled PTC-C4d staining, but were more predictive of peritubular capillaritis (PTC). Donor-specific antibodies (DSA), PTC-C4d, EC4d and PTC were analyzed in an independent longitudinal follow-up cohort (96 biopsies, 76 patients). Seventy-six samples were PTC and EC4d concordant, 11 positive and 65 negative, 7 PTC-EC4d+ and 13 PTC+EC4d-. EC4d levels were related to DSA occurrence. With ABMR defined by PTC and DSA, all apparently discordant patients, EC4d negative, were correctly reassigned comparing EC4d level curves with rejection kinetics, with positive EC4d samples predating biopsy or late biopsies compared with ABMR flare-ups. All EC4d-positive patients without PTC or DSA had permanent high EC4d levels unrelated to rejection. EC4d was more abundant in PTC-positive (mean = 108.5%± 3.4; n = 50) than PTC-negative samples (mean = 88.1%± 1.3; n= 96; p < 0.0001). Sensitivity, specificity, positive predictive value and negative predictive value of PTC-C4d and EC4d for PTC were, respectively, 75%, 79%; 64%, 76% (p < 0.05); 28%, 46% (p < 0.05) and 93%, 94%. Values were similar for DSA. A noninvasive blood test, EC4d, and particularly longitudinally monitoring EC4d levels, may increase surrogate ABMR testing options.

Combination of KIR and HLA gene variants augments the risk of developing birdshot chorioretinopathy in HLA-A*29-positive individuals.

Birdshot chorioretinopathy (BCR), a chronic ocular inflammatory disease with characteristic choroidal lymphocytic infiltrates, has been strongly associated with human leukocyte antigen (HLA)-A29. Although HLA-A29 occurs frequently in all populations, BCR affects only a small percentage of HLA-A29-positive Caucasians, indicating additional susceptibility factors for BCR. Discovery of HLA class I-specific killer cell immunoglobulin-like receptors (KIR) led to a series of epidemiological studies implicating KIR-HLA gene combinations in disease. Here, we characterized KIR-HLA pairs in BCR patients and controls carrying HLA-A*29 as well as controls lacking HLA-A*29. KIR-HLA pairs implicated for weak inhibition (KIR2DL2/3+HLA-C1 and KIR3DL1+HLA-Bw4(T80)) in combination with activating KIR genes associated with autoimmunity (KIR2DS2, 2DS3 and 2DS4) augment the risk of developing BCR in HLA-A*29-positive individuals. The reciprocal association of strong inhibitory pairs (KIR3DL1+HLA-Bw4(I80) and KIR2DL1+HLA-C2) in combination with those implicated in protection from infection (KIR3DS1+HLA-Bw4(I80) and KIR2DS1+HLA-C2) was observed in HLA-A*29-negative controls. These results suggest that a profound effect of KIR2DS2/S3/S4 in the absence of strong inhibition may enhance the activation of natural killer cells and T-cell subsets against intraocular self-antigens, thereby contributing to pathogenesis of BCR.

[Nano-biocaptures for research and diagnostics in inflammation diseases and cancer]. / Nano-biocapteurs pour la recherche et les diagnostics des maladies inflammatoires et du cancer.

As part of the ongoing search for ways to decrease the mortality of different pathological conditions related to cancer and inflammatory diseases, nanotechnologies currently under evaluation offer potentially attractive tools for innovative methodologies for early diagnosis, new bioimaging techniques and therapeutic strategies. Nano-tools can be employed for various functions, such as the detection of lesions at very early stages of disease development, extremely precise anatomical localization, or evaluation of the efficacy of medications specifically targeted against cells and pathological tissues. We have synthesized homogeneous CdSe/ZnS (core/shell) highly fluorescent nanocrystals (NC) detectable as individual nanoparticules with a routine fluorescent microscope. These NC are at least 10-fold brighter than the best organic fluorophores and at least 1000-fold more stable against photobleaching than AlexaFluor, for example. When conjugated with proteins, DNA or with drugs, NCs may be excited with the light of any wavelength from UV through visible spectral region providing a range of fluorescence colors depending on their diameter. These properties provide excellent perspectives for high through-put multiplexing and long-term tracking of labeled precursors for days or even weeks. We present here NC applications for ultrasensitive detection of p-glycoprotein, cytokeratins, LCA, Ki67, etc. both on the cellular level and in pathological human surgical specimens.

A simple method for accurate quantification of complement receptor 1 on erythrocytes preserved by fixing or freezing.

The mean number of complement receptor 1 (CR1) molecules on erythrocytes differs between normal individuals within the range of 100-1000 molecules per cell. In some disease states such as systemic lupus erythematosus (SLE), acquired immune deficiency syndrome (AIDS), insulin-dependent diabetes mellitus and malaria, erythrocyte CR1 levels are reduced and CR1 function may be impaired. Current methods for determining erythrocyte CR1 levels by flow cytometry require the use of freshly drawn blood samples because CR1 is lost from erythrocytes during storage. In order to facilitate field studies of associations between erythrocyte CR1 levels and disease, we have developed and validated an assay to quantify CR1 on both healthy and diseased erythrocytes that have been fixed in 5% formaldehyde or frozen in glycerol. These methods enable blood samples to be collected in areas lacking the facilities for flow cytometry and stored for later accurate quantification of CR1. Such procedures will be of particular benefit for future investigations of erythrocyte CR1 expression level and malaria susceptibility.

Unusual direct phycoerythrin labeling of B-cells from a splenic marginal zone lymphoma.

Flow cytometry is the most widely used method for lymphocyte subset characterization. Two types of antibodies, directly labeled with fluorochrome, are currently used for immunological diagnosis of B-cell lymphoproliferation: monoclonal antibodies against leukocyte differentiation antigens and polyclonal antibodies against immunoglobulins and light chains. In this study is described the case of a patient with an uncommon immunophenotyping of a B-cell lymphoproliferative disorder. B-cells from peripheral blood and from bone marrow reacted positively with all the tested phycoerythrin (PE)-conjugated antibodies, including the isotypic control. So we thought about a B-cell proliferation carrying a surface receptor recognizing PE: these B-cells were directly labeled with streptavidin-PE, indeed. Moreover, the immunodots from the patient were able to fix the streptavidin-PE. Finally, this unusual immunophenotyping was solved by using antibodies labeled with other fluorochromes than PE.

A complement receptor-1 polymorphism with high frequency in malaria endemic regions of Asia but not Africa.

Complement receptor-1 (CR1) is a ligand for rosette formation, a phenomenon associated with cerebral malaria (CM). Binding is dependent on erythrocyte CR1 copy number. In Caucasians, low CR1 expressors have two linked mutations. We determined the Q981H and HindIII RFLP distribution in differing population groups to ascertain a possible role in adaptive evolution. We examined 194 Caucasians, 180 Choctaw Indians, 93 Chinese-Taiwanese, 304 Cambodians, 89 Papua New Guineans (PNG) and 366 Africans. PCR/RFLP used HindIII for CR1 expression and BstNI for the Q981H mutation. DNA sequencing and pyrosequencing were performed to resolve inconclusive results. Gene frequencies for the L allele were 0.15 in Africans, 0.16 in Choctaws, 0.18 in Caucasians, 0.29 in Chinese-Taiwanese, 0.47 in Cambodians and 0.58 in PNG. Allelic frequency for 981H were 0.07 in Africans, 0.15 in Caucasians, 0.18 in Choctaws, 0.29 in Chinese-Taiwanese, 0.47 in Cambodians and 0.54 in PNG. The Q981H polymorphism correlates with the HindIII RFLP in most groups except West Africans and appears to be part of a low CR1 expression haplotype. The gene frequency for the haplotype is highest in the malaria-endemic areas of Asia, suggesting that this haplotype may have evolved because it protects from rosetting and CM.

New HLA-DRB1*07 allele identified from microlymphocytotoxicity/SSP discrepancies.

A HLA-DRB1*07 variant allele has been identified in a cadaver kidney donor. Serological typing using monoclonal antibodies detected HLA-DR4 and HLA-DR7. HLA class II DNA typing using sequence-specific primer (PCR-SSP) polymerase chain reaction only detected DRB1*04, while sequence-specific oligonucleotide (PCR-SSO) polymerase chain reaction confirmed the presence of both DRB1*04 and DRB1*07 alleles, although two extra reactions were also found. Exon 2 of the HLA-DRB1*07 was isolated using allele-specific PCR, then cloned and sequenced. Four mutations, at positions 170 (T --> C), 171 (C --> T), 174 (C --> G), and 179 (C --> A), were observed. These mutations changed codons 57 and 60 (V --> A; S --> Y, respectively). This amino acid sequence at position 56-61 is only found in DRB1*0811.

Serotonin transporter promoter variants in autism: functional effects and relationship to platelet hyperserotonemia.

The well-replicated platelet hyperserotonemia of autism has stimulated interest in serotonin (5-HT) in autism. We have examined the effects of the serotonin transporter gene (5-HTT, locus SLC6A4) promoter polymorphism (5-HTTLPR) on platelet 5-HT physiology in autism. Platelet 5-HT uptake rates and affinities (V(max) and K(m)), uptake site densities (B(max)) and 5-HT levels were examined in 31 French individuals with autism genotyped with respect to the 5-HTTLPR. Platelet 5-HT uptake and 5-HT levels were measured using HPLC; uptake sites were determined by radioligand binding. A 1.5-fold increased rate (V(max)) of platelet 5-HT uptake was observed in ll genotype individuals compared to those with ls and ss genotypes (Mann- Whitney U-test, P = 0.022). However, no significant relationship was observed between genotype and uptake site density (U-test, P = 0.51). Although median levels of platelet 5-HT in platelet-rich plasma were higher in the ll group, only trend level significance was observed (U-test, P= 0.069); platelet 5-HT content measured in whole blood was similar across genotypes. Uptake rates were well correlated with B(max) values (r = 0.66, P = 0.002); correlations between uptake and platelet 5-HT levels and between B(max) values and 5-HT levels were somewhat lower. While 5-HTTLPR alleles had an appreciable effect on platelet 5-HT uptake rates, effects on 5-HT levels and uptake site density were smaller or absent. Based on these preliminary data and prior studies of allele frequencies, we conclude that the 5-HTTLPR is not a major determinant of the group mean platelet serotonin elevation seen in autism. However, a role for increased uptake in the hyperserotonemia of autism can not be ruled out. In addition, it appears that studies of platelet 5-HT measures in autism and other disorders should take account of the effects of 5-HTTLPR genotype on 5-HT uptake